Ebook November 2017 by Clinics

Volume 72 Number 11 - November/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(11)649–717

EDITORIAL

Sudden unexpected death in Parkinson’s disease (SUDPAR): a review of publications since the decade of the brain Fulvio A. Scorza, Andrea C. do Carmo, Ana C. Fiorini, Mariana B. Nejm, Carla A. Scorza, Josef Finsterer, Henrique B. Ferraz . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649

CLINICAL SCIENCE

Role of T. cruzi exposure in the pattern of T cell cytokines among chronically infected HIV and Chagas disease patients Tania Regina Tozetto-Mendoza, Dewton de Moraes Vasconcelos, Karim Yaqub Ibrahim, Ana Marli Christovam Sartori, Rita C. Bezerra, Vera Lúcia Teixeira de Freitas, Maria Aparecida Shikanai-Yasuda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 652

Assessment of the quality of life, muscle strength, and dynamic balance of elderly Kendo players Dário Lucas Costa de Mendonc¸a, Angelica Castilho Alonso, Júlia Maria D’Andrea Greve, Luiz Eugênio Garcez-Leme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661

Cardiorespiratory performance of coronary artery disease patients on land versus underwater treadmill tests: a comparative study Mauricio Koprowski Garcia, Limanara Rizzo, Paulo Yazbek-Júnior, Daniela Yutiyama, Fabiola Jomar da Silva, Denise Matheus, Luiz Eduardo Mastrocolla, Eduardo Massad . . . . . . . . . . . . . . . . . . . . . . 667

Impact of the delay to start treatment in patients with lung cancer treated in a densely populated area of Brazil Fernando Conrado Abrao, Igor Renato Louro Bruno de Abreu, Roberto Odebrecht Rocha, Felipe Dourado Munhoz, João Henrique Godoy Rodrigues, Riad Naim Younes . . . . . . . . . . . . . . . . . . . . . . . . . . . 675

Dental status, oral prosthesis and chewing ability in an adult and elderly population in southern Brazil Alexandre Baumgarten, Jeanne Gabriele Schmidt, Rafaela Soares Rech, Juliana Balbinot Hilgert, Bárbara Niegia Garcia de Goulart . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681

Prognostic factors of the short-term outcomes of patients with hepatitis B virus-associated acute-on-chronic liver failure Qing Lei, Kangjian Ao, Yinhua Zhang, Deqiang Ma, Deping Ding, Changzheng Ke, Yue Chen, Jie Luo, Zhongji Meng . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 686

Videoﬂuoroscopic analysis of different volumes of liquid bolus swallowing in healthy individuals: comparison between height and sex Marcia Regina Kfouri Bernardi Regueiro, Weslania Viviane Nascimento, Luana Casari Parreira, Roberto Oliveira Dantas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693

A novel body mass index reference range - an observational study Sirlei Siani Morais, Mirena Ide, Andrea Moreno Morgan, Fernanda Garanhani Surita . . . . . . . . . . . . . . . . . . . . . . . 698

BASIC RESEARCH

Peri-implant assessment via cone beam computed tomography and digital periapical radiography: an ex vivo study Nicolau Silveira-Neto, Mateus Ericson Flores, João Paulo De Carli, Max Dória Costa, Felipe de Souza Matos, Luiz Renato Paranhos, Maria Salete Sandini Linden . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708

RAPID COMMUNICATION

Evidence of progenitor cells in the adult human cochlea: sphere formation and identiﬁcation of ABCG2 Milene Massucci-Bissoli, Karina Lezirovitz, Jeanne Oiticica, Ricardo Ferreira Bento . . . . . . . . . . . . . . . . . . . . . . . . 714

EDITORIAL

Sudden unexpected death in Parkinson’s disease (SUDPAR): a review of publications since the decade of the brain Fulvio A. Scorza,I,* Andrea C. do Carmo,II Ana C. Fiorini,III,IV Mariana B. Nejm,I Carla A. Scorza,I Josef Finsterer,V Henrique B. FerrazVI I Disciplina de Neurociencia, Escola Paulista de Medicina, Universidade Federal de Sao Paulo (EPM/UNIFESP), Sao Paulo, SP, BR. II Biblioteca do Campus Sao Paulo, Universidade Federal de Sao Paulo, Sao Paulo, SP, BR. III Programa de Estudos Pos-Graduados em Fonoaudiologia, Pontificia Universidade Catolica de Sao Paulo (PUC-SP), Sao Paulo, SP, BR. IV Departamento de Fonoaudiologia, Escola Paulista de Medicina, Universidade Federal de Sao Paulo (EPM/ UNIFESP), Sao Paulo, SP, BR. V Department of Neurology, Krankenanstalt Rudolfstiftung, Vienna, Austria. VI Departamento de Neurologia, Escola Paulista de Medicina, Universidade Federal de Sao Paulo (EPM/UNIFESP, Sao Paulo, SP, BR.

*Corresponding author. E-mail: acfiorini@pucsp.br

Although increasing insight into the course and pathomechanisms of PD has been achieved, including risk factors that increase mortality rates, much of the disease remains a mystery (5,10-12,19,20). Importantly, previous studies suggest that PD is not a benign condition and is occasionally associated with sudden unexpected death in PD (SUDPAR) (21,22). The causes of SUDPAR are unknown, but cardiovascular risk factors (B60% of people with PD have cardiovascular abnormalities) may play an important role (22-25). Notably, one practical problem in studying the risk factors, mechanisms and prevention of SUDPAR is that the condition is relatively uncommon. Therefore, the development of translational studies in PD patients who carry a high risk of premature mortality would be of interest to researchers (21,22). Matsumoto et al. reviewed clinical data and the causes of death of 16 PD patients who underwent postmortem examinations (23). Their study revealed that four of 16 PD patients died of SUDPAR without any satisfactory causes detected by autopsy, making SUDPAR the second most common cause of death among the population evaluated (23). Based on these results, the authors proposed that, although bias is derived from postmortem examinations, a non-negligible number of PD patients die of SUDPAR (23). A substantial increase has occurred in the number of scientific publications on PD during the last three decades (5,10). However, how many of these publications were studies on mortality in PD? Our research group performed a descriptive review of the published literature on mortality in PD research since the ‘‘Decade of the Brain’’ was declared. We found 143,006 articles associated exclusively with PD and 1,982 scientific papers related to mortality in PD (Figure 1). Approximately 2% of the research articles published in the scientific community since the ‘‘Decade of the Brain’’ was declared are related to mortality or to aspects associated with SUDPAR. These studies indicate that life expectancy is reduced in patients with PD compared to the general population, irrespective of comorbidities, suggesting that specific characteristics of PD are responsible for the increased

The past 30 years have been an important period in Parkinson’s disease (PD) research. PD is the second most common neurodegenerative disorder, after Alzheimer’s disease, and affects 2-3% of the population X65 years of age. Unfortunately, many studies have shown that individuals with PD have a higher risk of mortality than the general population, and sudden unexpected death in Parkinson’s disease (SUDPAR), an unusual but fatal event, also occurs. SUDPAR is a poorly described phenomenon, and translational studies should be considered and evaluated to establish new frontiers in the field of SUDPAR research. Since the ‘‘Decade of the Brain’’ was declared, several neuropsychiatric diseases, including PD, are now seen as specific diseases with explicable causes and effective measures of prevention, treatment and rehabilitation (1-4). PD is one the most common neurodegenerative diseases and is characterized by tremors, muscular rigidity, slowed movements and postural imbalance resulting from progressive neuronal loss in the substantia nigra; this neuronal loss causes striatal dopamine deficits and intracellular inclusions containing aggregates of a-synuclein (5-9). Despite rapid advances in our understanding of PD at various levels (from cellular mechanisms to more sophisticated forms of treatment) (10), neuroscientists cannot comprehensively assess the risk of PD-related death. Unfortunately, PD patients have a higher risk of mortality than the general population (11-18). In fact, a recent 38-year follow-up study demonstrated that the mortality of PD patients does not increase during the first decade after disease onset but increases thereafter, eventually reaching twice the level of that of the general population (5,19). The main causes of death for these patients were pneumonia and cerebrovascular and cardiovascular diseases (19).

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(11)01

649

Sudden Death in Parkinson’s disease Scorza FA et al.

CLINICS 2017;72(11):649-651

Figure 1 - Distribution of publications (log scale) on ‘‘Parkinson’s’’ and ‘‘Parkinson’s and Mortality’’ from 1988 to 2016, in intervals of five years. Publications related exclusively to PD are shown in blue (143,006 articles). Publications related to mortality in PD are shown in orange (1,982 articles). Data were identified through searches of MEDLINE, SCOPUS and LILACS.

a close clinical convergence. Whenever necessary, strategies for routine cardiovascular screening (e.g., ECG, Holter-monitoring and echocardiography) should be undertaken to further reduce the likelihood of SUDPAR. Finally, to better understand the incidence, potential risk factors, principal mechanisms and preventive strategies of SUDPAR, well-designed clinical studies and basic research in validated animal models should be conducted.

mortality (13,14,26). Over the past 30 years, several longterm clinical follow-up studies have identified that age at onset, motor severity, dementia and psychotic symptoms are independent risk factors of mortality in PD patients (26). However, information on SUDPAR remains insufficient. What is SUDPAR? The first step in the process of characterizing any community-wide disease condition is the definition of the problem (27). SUDPAR is a category of death in individuals with PD and not a condition of the disorder. As has been widely discussed in epilepsy (28), SUDPAR is particularly difficult to investigate in translational studies due to its rarity and the insufficiency of postmortem examinations (21-23,28-30). SUDPAR could be defined simply as the unexpected death of a patient with PD without any satisfactory causes of death determined by autopsy. After this definition was proposed, several studies evaluated the effects of potential risk factors that could increase the probability of mortality in PD patients, such as age at onset, duration of PD, gender, motor severity and drug treatment (polypharmacy) (5,20-22,25,26,31). These risk factors may be directly related to the occurrence of SUDPAR, but further research is required to establish their precise roles as potential SUDPAR risk factors. Moreover, the mechanisms of SUDPAR remain unknown. Human and experimental research suggest that cardiac abnormalities and autonomic dysfunction play key roles in SUDPAR (21-23,31-33). While doubts still exist and some speculative proposals have been described (21-22,33), the best approach is to try to prevent possible cardiac abnormalities in PD patients. Accordingly, appropriate global cardiovascular assessments of individuals with PD should be performed. First, a comprehensive clinical history including the evaluation of a possible family history of SUDPAR should be obtained. Second, a full physical examination is fundamental. Third, individuals with PD should reduce cardiovascular risk factors, such as cigarette smoking, obesity, high blood pressure, hyperglycemia, hyperlipidemia, and alcohol intake, with the aim of minimizing heart disease and fatal cardiovascular events. Fourth, neurologists and cardiologists should develop

’ ACKNOWLEDGMENTS This study was supported by the following grants: FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo); CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico); CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and FAPESP/CNPq/ MCT (Instituto Nacional de Neurociência Translacional).

’ AUTHOR CONTRIBUTIONS Scorza FA critically discussed and wrote the manuscript. do Carmo AC acquired and analyzed data, and critically discussed and wrote the manuscript. Fiorini AC and Nejm MB analyzed data and critically discussed and wrote the manuscript. Scorza CA, Finsterer J and Ferraz HB critically discussed and wrote the manuscript.

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Role of T. cruzi exposure in the pattern of T cell cytokines among chronically infected HIV and Chagas disease patients Tania Regina Tozetto-Mendoza,I,II,# Dewton de Moraes Vasconcelos,III,# Karim Yaqub Ibrahim,IV Ana Marli Christovam Sartori,IV Rita C. Bezerra,VI Vera Lu´cia Teixeira de Freitas,I,V Maria Aparecida Shikanai-YasudaI,V,* I Laboratorio de Imunologia (LIM 48), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Laboratorio de Virologia (LIM 52), Universidade de Sao Paulo, Instituto de Medicina Tropical, Sao Paulo, SP, BR. III Laboratorio Dermatologia e Imunodeficiencias (LIM-56), Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. IV Divisao de Doencas Infecciosas e Parasitarias, Hospital das Clinicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. V Departamento de Doencas Infecciosas e Parasitarias, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR. VI Laboratorio de Parasitologia (LIM 46), Hospital das Clı´nicas HCFMUSP, Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVES: The impact of Chagas disease (CD) in HIV-infected patients is relevant throughout the world. In fact, the characterization of the adaptive immune response in the context of co-infection is important for predicting the need for interventions in areas in which HIV and Chagas disease co-exist. METHODS: We described and compared the frequency of cytokine-producing T cells stimulated with soluble antigen of Trypanosoma cruzi (T. cruzi) using a cytometric assay for the following groups: individuals with chronic Chagas disease (CHR, n=10), those with Chagas disease and HIV infection (CO, n=11), those with only HIV (HIV, n=14) and healthy individuals (C, n=15). RESULTS: We found 1) a constitutively lower frequency of IL-2+ and IFN-g+ T cells in the CHR group compared with the HIV, CO and healthy groups; 2) a suppressive activity of soluble T. cruzi antigen, which down-regulated IL-2+CD4+ and IFN-g+CD4+ phenotypes, notably in the healthy group; 3) a down-regulation of inflammatory cytokines on CD8+ T cells in the indeterminate form of Chagas disease; and 4) a significant increase in IL-10 +CD8+ cells distinguishing the indeterminate form from the cardiac/digestive form of Chagas disease, even in the presence of HIV infection. CONCLUSIONS: Taken together, our data suggest the presence of an immunoregulatory response in chronic Chagas disease, which seems to be driven by T. cruzi antigens. Our findings provide new insights into immunotherapeutic strategies for people living with HIV/AIDS and Chagas disease. KEYWORDS: Intracellular Cytokines; Chagas Disease; HIV; Trypomastigote Antigen; T Cells. Tozetto-Mendoza TR, Vasconcelos DM, Ibrahim KY, Sartori AM, Bezerra RC, de Freitas VL, et al. Role of T. cruzi exposure in the pattern of T cell cytokines among chronically infected HIV and Chagas disease patients. Clinics. 2017;72(11):652-660 Received for publication on March 6, 2017; First review completed on June 23, 2017; Accepted for publication on July 19, 2017 *Corresponding author. E-mail: masyasuda@yahoo.com.br #

These authors contributed equally to this work.

’ INTRODUCTION

acute cases and outbreaks (1). Migration from rural areas has made chronic CD primarily an urban disease in Latin America and the United States (1-6). Transmission of CD through blood transfusion, blood by-products, or organ transplantation is currently a serious problem in non-endemic areas. At least 5 to 6 million chronically infected individuals live in endemic and non-endemic countries, and the disease continues to represent a health threat around the world (6). Acute CD is characterized by alterations in the mononuclear phagocytic system, causing lymphadenopathies and, less frequently, severe myocarditis or meningoencephalitis. Additionally, although most chronically infected individuals are asymptomatic, approximately 30 to 40% develop recognized cardiomyopathy or gastrointestinal tract disorders (7). Reactivation of CD manifests as a febrile syndrome with meningoencephalitis and/or myocarditis, which is also

Chagas disease (CD), caused by the protozoan Trypanosoma cruzi (T. cruzi), which is transmitted to humans by a vector (reduviid bugs), is currently one of the most important neglected diseases in Latin America. After the control of transmission by Triatoma infestans, the main vector in Brazil, the most important route is oral transmission associated with

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of Clinics of Infectious and Parasitic Diseases of the Hospital das Clínicas, Faculdade de Medicina, University of São Paulo. HIV infection was diagnosed with ELISA and confirmed with Western blotting. CD4+ and CD8+ T cell counts were measured by flow cytometry, and HIV viral load was determined by polymerase chain reaction (PCR). All HIVinfected individuals received Highly Active Antiretroviral Therapy (HAART), according to the national guidelines for antiretroviral therapy (15). For CD diagnosis (the CHR group), seropositivity in at least two of three tests for T. cruzi infection (i.e., ELISA, indirect immunofluorescence assay, and indirect haemagglutination assay) was required. The clinical forms of CD were classified according to Sartori et al. 2007 (22): (1) ‘‘Indeterminate form’’ (IND), meaning no symptoms or signs and normal results for electrocardiogram (ECG), chest X-ray, and oesophagography. (2) ‘‘Non-typical cardiopathy’’ or ‘‘atypical manifestations’’ (AT), meaning ECG changes possibly related to conditions other than CD. (3) ‘‘Typical cardiopathic form’’ with ECG changes considered typical of CD, including sinus bradycardia o50 beats/min, right bundle-branch block, second-degree atrioventricular block, and/or complex ventricular arrhythmia. (4) ‘‘Digestive form’’, meaning megaoesophagus or megacolon. (5) ‘‘Typical digestive and cardiopathy’’ (TDC), meaning typical cardiac and/or digestive manifestation. Xenodiagnosis (23) and blood culture (24) were requested according to medical orientation and performed by the Laboratory of Parasitology of the Clinics Hospital. Blood culture and/or xenodiagnosis data were used to confirm the presence or absence of parasites (T. cruzi) or reactivation of infection by T. cruzi at the time of this study. Qualitative PCR was performed using primer pairs for S35 and S36 kinetoplast sequences (25). Qualitative PCR was also performed using TCZ3/TCZ4 microsatellite sequences (26). Therefore, we analysed four groups. There were 10 individuals with chronic CD and not infected with HIV (CHR group), 11 with chronic CD and HIV infection (CO group), 14 with HIV infection without CD (HIV group) and 15 healthy individuals (C group). The C group consisted of individuals reporting the absence of CD, HIV diagnosis or any other chronic or acute illnesses, with CD4+ and CD8+ levels above those associated with susceptibility to opportunistic diseases (4500 cells/mm3) and aged o50 years old. We assessed the frequencies of CD4+ or CD8+ T cells staining positive for IL-2, IFN-g, IL-4, and IL-10 after being incubated in vitro with SAg and/or mitogens (phorbol and ionomycin), according to the methods described below. Concerning the available data on the clinical form of CD in individuals with or without HIV infection, we compared two sub-groups of CD: typical digestive and/or cardiac manifestation (TDC) (n=8) and indeterminate form (IND) (n=8). Two patients with an atypical clinical (AT) form of CD and three with an unknown or non-identified (NI) form of CD were excluded from these analyses.

associated with HIV infection and other immunodeficiency states such as haematological malignancies, bone marrow, kidney, or heart transplantation, and corticosteroid therapy (8-11). Reactivation of CD in AIDS patients has been observed in p20% of co-infected patients and has sometimes been reported as the first opportunistic infection (12). According to Almeida et al. 2011 (13), the overall mortality rate of HIV patients was 30%, and mortality occurred in 73% of the cases in which there was reactivation of CD. The frequency of individuals co-infected with HIV and T. cruzi was estimated at 1.3% in a Brazilian study with a macro-regional approach (14). The impact of co-infection is extremely relevant, as approximately 1.5 million individuals with HIV/AIDS live in Latin America and approximately 21,420 HIV/T. cruzi co-infected patients are estimated to live in this area (15). Immunoregulatory mechanisms may influence the pathogenesis and clinical evolution of CD (16). Because CD and HIV infection are both associated with T cell responses and disturbances of cytokine networks (17, 18), the characterization of cytokine-secreting T cells is particularly relevant to improving our understanding of the immunopathogenesis of CD and to controlling concomitant intracellular infections in AIDS and other immunosuppressive conditions. A study of the differential regulation of Th1 and Th2 responses in HIV infection showed that decreased secretion of type-1 cytokines, such as IL-2 and IFN-g, was associated with a higher susceptibility to opportunistic infections (19). Conversely, previous studies of the pathogenesis and clinical evolution of CD have reported higher IL-4/IFN-g ratios in patients with HIV/Chagas disease as well as the preferential involvement of inflammatory cytokines and activated T cells (18, 20). However, it is unclear whether the presence of HIV and T. cruzi co-infection modifies this mechanism in humans. Recent studies have demonstrated the impact of a specific antigenic stimulus on the course of a chronic infection in mice, as seen in the association between an HIV vaccine and helminthic infection (21). Accordingly, the characterization of the adaptive immune response either in mouse models or in human infections is relevant to interpreting or predicting therapeutic interventions in endemic areas where HIV and other infections co-exist. This study aimed to describe and compare the profiles of cytokine-producing T cells from individuals with chronic Chagas disease and/or HIV infection with those from healthy individuals using a cytometric assay, which detects the intracellular accumulation of cytokines in CD4+ and CD8+ T lymphocytes stimulated with soluble trypomastigote antigens and mitogens.

’ MATERIALS AND METHODS Ethics Statement The Human Research Ethics Committee of the Hospital das Clínicas, Faculdade de Medicina, University of São Paulo approved the research protocol (CAPPesp 010/95-B). A signed informed consent form was obtained from all 50 participants (35 patients diagnosed with CD and/or HIV infection and 15 healthy individuals) for the period of 2001-2005 to participate in the present cross-sectional study based on convenience sampling.

Cell Preparation and In Vitro Stimulation with Soluble Antigen of T. cruzi Trypomastigote (SAg) Samples of peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood (30 mL) using density gradient sedimentation (1.077 g/mL) in accordance with the recommendations of the manufacturer (Ficoll-Paque Amersham Bioscience, Piscataway, NJ, USA). The cells were then washed three times with sterile phosphate-buffered

Study Groups and Methods We enrolled 35 patients diagnosed with chronic CD and/or HIV infection who attended the Outpatient Clinic, Division

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Cytokines in Chagas disease and HIV infection Tozetto-Mendoza TR et al.

Figure 1 - Flow cytometric evaluation. (A) T. cruzi-stimulated cells. Lymphocytes in the R1 region based on the SSC/FSC (Side Scatter/Forward Scatter) ratio; (E) CD3 surface marker staining on lymphocytes gated in R1; (B, C, D, F, G, H) CD8 surface- and intracellular IFN-g-stained cells were simultaneously gated in R1 (lymphocytes) and R2 (CD3+ cells). R3 and R5 regions represent CD8+ and CD8- (i.e.: CD4+) cells negative for IFN-g, respectively; R4 and R6 represent CD8+IFN-g+ and CD4+IFN-g+ cells, respectively. Peripheral blood mononuclear cells were cultured for 72 h without T. cruzi antigen (B, C, D) and with T. cruzi antigen (F, G, H) and subsequently exposed to PMA, ionomycin, and brefeldin A (BFA) for the last 4 h. For each analysis, approximately 50,000 events were acquired on a FACSCalibur flow cytometer. (B) Monocyte gate strategy. Flow cytometric acquisition and subsequent analysis were manually performed within a monocyte gate defined for each sample using CellQuest (BD Bioscience, Immunocytometry System) based on a higher FSC/SSC ratio and negative expression of CD3 and CD8 (R7 region) and are displayed as histograms showing cytokines (PE) (R8 region).

cell to produce these cytokines. It has been shown that following stimulation of lymphocytes with PMA and ionomycin, a rapid down-regulation of CD4 molecules on the surface of lymphocytes occurs (28). In our hands, a rapid decrease in CD4 occurred within 4 hours after stimulation. Therefore, a negative-gating strategy (among CD3+ T cells, CD8- cells were selected) was used to measure the intracellular cytokine expression in CD3+CD4+ cells. To normalize the comparison of inter-assay results, each assay analysed PBMCs from individuals in the four groups, with at least one individual of each group (C, CO, HIV and CHR) measured simultaneously.

saline (PBS) without Mg++/Ca++ (Gibco BRL, UK) and re-suspended at 2.5 x 106 cells/mL in RPMI 1640 (Gibco BRL, UK) supplemented with 10% heat inactivated foetal calf serum (HyClone, Logan, Utah, USA), 2 mM L-glutamine (Gibco BRL, UK) and antibiotics, according to Sousa e Victorino, (1998) (27) for in vitro stimulation and immunofluorescence staining of freshly isolated cells. The soluble antigen obtained by complete disruption of 1.0 x 106 parasites/mL (10 cycles of freezing and thawing at the trypomastigote stage) was kindly provided by Dr. Ises de Almeida Abrahamsohn (Department of Immunology of the Institute of Biomedical Sciences of the University of SĂŁo Paulo, SP, Brazil). Two different antigenic stimuli were used to stimulate cells in vitro, according to De Barros-Mazon et al. (2004) (20), but with some modifications. One set of cells was stimulated with specific soluble trypomastigote antigen (SAg) for 72 hours followed by brefeldin A (BFA, Sigma Chemical) at a final concentration of 10 mg/ml and phorbol 12-myristate 13-acetate (PMA, Sigma Chemical) + ionomycin (I, Sigma Chemical) at a final concentration of 50 ng/mL and 500 ng/mL, respectively, for the last 4 hours. In addition, another sample of cells was subjected to the same protocol with only PMA+I+ BFA and not SAg to measure the potential capability of each

Monoclonal Antibodies (MoAbs) for Intracellular and Cell Surface Staining The following phycoerythrin-conjugated anti-human cytokines and corresponding isotype controls were used for each antibody and each patient: anti-IL-2, clone M062626; antiIFN-g, clone M064882; anti-IL-4, clone M064843; anti-IL-10, clone M063277; rat IgG2a, clone M063752; and mouse IgG1, clone M056389. Surface staining was performed with anti-CD3 phycoerythrin-cyanin 5 (PE-Cy5), clone M047602, and anti-CD8 fluorescein isothiocyanate (FITC), clone M055034. All antibodies were purchased from Pharmingen (San Diego, CA).

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Immunofluorescence Staining and Flow Cytometric Analyses

The median age distribution was 30, 40, 47 and 42 years old for the C, HIV, CO and CHR groups, respectively. The proportion of male participants was lower in the CHR group (30.0%) than in the CO group (54.5%) and HIV group (78.6%), but the differences did not reach statistical significance among these groups (Kruskal Wallis test, p=0.9639). There was an equal proportion of females in the CHR group and C group (70-73%). Moreover, there was no difference in the CD4+ cell count among the HIV, CO and CHR groups (Kruskal Wallis test, p=0.0921) (Table 1A). The distribution of CD sub-groups and the mean CD4+ cell count among individuals with positive (n=4) and negative parasitaemia (n=11) detected by haemoculture, xenodiagnosis and PCR were also presented. PCR analysis for parasite DNA fragments was positive in two chagasic individuals with HIV infection (CO group) of eight available plasma samples. The CD4+ cell count tended to be higher in individuals with negative parasitaemia than with positive parasitaemia, but without statistical significance (Table 1B).

Cells were washed in PBS with 0.1% sodium azide (PBSazide). First, the Fc receptors of the cells were blocked with 2 mg of human IVIg (50 mg/ml) and incubated for 20 minutes at room temperature to decrease non-specific staining. Next, the cells were washed in PBS-azide and re-suspended in PBS (Gibco, without Ca+ and Mg+) with 1% bovine serum albumin (BSA, Sigma) and 0.1% sodium azide (Azide) (staining buffer). After surface staining with anti-CD8 and antiCD3 MoAbs, the cells were washed with staining buffer and fixed with PBS-1% paraformaldehyde (fixation buffer). These steps were performed at 4oC in the dark. Subsequently, the cells were permeabilized with PBS with 1% BSA and 0.5% saponin (permeabilization buffer). Intracellular staining was performed by incubation with anti-IL-2, anti-IL-4, anti-IL-10, and anti-IFN-g MoAbs or respective isotype controls for 30 minutes at room temperature in the dark. Next, the cells were washed and re-suspended in PBS-azide for flow cytometric analysis. For each analysis, approximately 50,000 events were acquired on a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). Flow cytometric acquisition was performed within a lymphocyte gate defined for each sample using CellQuest (BD Bioscience, Immunocytometry System) and displayed as dot plots showing cytokines (PE) versus CD3 (PE-Cy5) and CD8 (FITC) fluorescence in multiple dot plots. The CD8 dot plot was analysed for double dependence and CD3 expression on an FSC-SSC lymphocyte gate (Figure 1, Figure 1A). The monocyte gate was determined by FSC-SSC and exclusion of CD3+ and CD8+ populations (Figure 1, Figure 1 B).

Table 1B - CD4 cells/mm3 in 15 Chagas disease individuals (ALL) according to the presence of parasitaemia (detected by PCR, haemoculture and xenodiagnoses).

Statistical Analysis For comparisons between two groups, the Mann-Whitney test was used. For within-group comparisons, the Wilcoxon test was used. For comparisons among three groups (HIV, C and CHR), the Kruskal Wallis test was used. Statistical significance was set at 5% (po0.05). The Spearman correlation coefficient was used to measure bivariate associations of the proportion of cytokines.

ALL Chagas disease

Positive parasitemia

Negative parasitemia

Disease form (Number of Patients) CO and IND CO and TDC CHR and IND CHR and TDC CHR and NI HIV viral load (copies/mL) Mean (SD) Indetectable (%) Mean (SD) CD4 cells/mm3 Total Number of Patients

1 1 1 1 0

3 4 2 1 1

3.6 (3.5)a 25.0 361 (166)c 4

4.3 (4.1)b 66.7 680 (278)c 11

CD=Chagas disease; C=healthy controls; HIV=HIV infection; CO=co-infected with Chagas disease and HIV; CHR=chronic Chagas disease; TDC=typical digestive and/or cardiac involvement; IND=indeterminate form of CD; AT=atypical form of CD; NI=not identified; (SD)=standard deviation; n=number of patients. a two with available HIV viral load data. b six with available HIV viral load data. c p40.05 between CD4+ cells of positive and negative parasitaemia patients (Mann-Whitney test).

â&#x20AC;&#x2122; RESULTS Clinical and Laboratory Data of the Study Groups Data regarding the clinical groups at the time of enrolling in the study are shown in Table 1.

Table 1A - Study groups according to CD4+ cell count, age, sex and sub-groups of Chagas disease. Variables CD4+ count /cells/mm3 Age Female Subgroups of CD TDC IND AT NI

groups

HIV

CHR

n median (min-max) years (median) %

15 961 (933-988) 30 73.0

14 475 (243-870) 40 21.4

11 505 (218-837) 47 45.5

10 855 (551-1198) 42 70.0

n n n n

NA NA NA NA

4 4 2 1

4 4 0 2

min=minimum; max=maximum; n=number of individuals; CD=Chagas disease; C=healthy controls; HIV=HIV infection; CO=co-infected with Chagas disease and HIV; CHR=chronic Chagas disease; TDC=typical digestive and/or cardiac involvement; IND=indeterminate form of CD; AT=atypical form of CD; NI=not identified; NA=not applied; (SD)=standard deviation; n=number of patients.

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Figure 2 - Comparison of the potential capability of T cells to produce cytokines. (A-D) Proportion of cytokines on CD4+ T cells. (E-H) Proportion of cytokines on T CD8+ T cells. C = healthy controls; HIV = HIV infection; CO = co-infected with Chagas disease and HIV; CHR = chronic Chagas disease. IL-2+CD4+ phenotype: a statistically significant difference was observed between the CHR and HIV groups (Mann-Whitney test, p=0.0322), the CHR and C groups (Mann-Whitney test, p=0.0011), and the CHR and CO groups (Mann-Whitney test, p=0.0049). IL-2+CD8+ phenotype: a statistically significant difference was observed between the CHR and HIV groups (Mann-Whitney test, p=0.0441) and between the CHR and C groups (Mann-Whitney test, p=0.0436). IFN-g+CD4+ phenotype: a statistically significant difference was observed between the CHR and C groups (Mann-Whitney test, p=0.0101) and between the CHR and CO groups (Mann-Whitney test, p=0.0153). No statistically significant differences were observed in relation to the other phenotypes.

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Figure 3 - Comparison of the proportion of cytokines on T cells with and without SAg stimulus. (A). IL-2+CD4+. (B) IFN-c+CD4+. The asterisk (*) represents the condition in which cells were stimulated with soluble T. cruzi antigen. The difference between nonstimulated and stimulated* cells was significant for IL-2+CD4+ (Wilcoxon test, p=0.0093) and IFN-g+CD4+ T cells (Wilcoxon test, p=0.048). No statistically significant differences were observed in relation to the other phenotypes.

Comparison of the Phenotype Frequency of Cytokines on CD4+ and CD8+ T cells from the C, HIV, CO, and CHR Groups

co-infected with HIV (ALL and ALL*, Mann-Whitney test, p=0.0317, Figure 4). We also found a negative correlation between IFN-g and IL-10 (p=0.0390; r=-0.6691) when the source of cytokines was CD8+ T cells derived from chronic chagasic patients.

In the Absence of SAg Antigen. The potential capacity of production of IL-2+ and IFN-g+ Tcells in the CHR group was low, as determined by comparing the CHR group with each other group (C, HIV, CO) and shown in Figure 2 (Figure 2A, 2B, 2E). No significant differences were found in other cellular phenotypes (Figure 2C, 2D, 2F, 2G, 2H).

â&#x20AC;&#x2122; DISCUSSION We have described the profile of intracellular cytokine production in T cells from HIV-infected and/or chagasic individuals by focusing on three important aspects, showing that T. cruzi exposure played a pivotal role in the downregulation of inflammatory cytokine-producing T cells in human infections. First, we showed that the potential capability of T cells to produce IL-2 and IFN-g was lower in individuals with chronic CD compared with each of the other groups analysed (HIV, CO or C). IL-2 has been described as a prototypic Th1 cytokine, being essential for the differentiation and activation of regulatory T cells, and as noted here, the decreased IL-2 production in chronic CD seems to suggest that an adaptive activation mechanism is triggered by prior exposure to T. cruzi infection (Figure 2, Figure 2A, 2B and 2E). The decreased inflammatory profile could be associated with the pathogenesis of cardiac and gastrointestinal diseases, since the immunoregulatory features seem to be linked to the indeterminate form of CD (29, 30). Second, we observed in vitro an immunomodulatory effect of SAg, mainly expressed in cells from healthy individuals. In fact, a significant down-regulation of IL-2+CD4+ and IFN-g+CD4+ cells was observed in healthy individuals (group C), who reported the absence of acute or chronic disease and did not belong to the risk group exposed to T. cruzi or HIV infection (Figure 3, Figure 3A, Figure 3B). We observed the absence of this effect in the CHR group, as these cell phenotypes were already low at background level in these patients. Corroborating our data, other protocols based either on soluble antigen or on cell cultures stimulated by living trypomastigotes suggest that this effect of the HIVT. cruzi interaction on cytokine secretion is parasite driven (31, 32). By contrast, protocols based on cellular stimulation with epimastigotes or recombinant B13 antigen do not seem

In the Presence of In Vitro SAg Stimulation. When we compared cells stimulated and non-stimulated with SAg from healthy individuals (group C) in particular, we observed a reduced proportion of IL-2+CD4+ and IFN-g+CD4+ cells in the presence of SAg, as shown in Figure 3 (Figure 3A, Figure 3B). We found no significant differences when we compared other T cell phenotypes from the CO, CHR and HIV groups in the presence or absence of SAg.

Comparison between the Indeterminate Form and Typical Clinical Manifestation of CD The results indicated a differential frequency of cell phenotypes between the IND and TDC sub-groups of CD (n=16), as shown in Figure 4. In Both the TDC (n=8) and IND (n=8) sub-groups, half of the patients were HIV-infected patients with low or undetectable viral load. In addition, six positive parasitological tests were detected in the sub-groups of CD. In the absence of SAg, the frequencies of IL-2+CD8+ and IL-10+CD8+ phenotypes were higher in sub-group IND than in sub-group TDC (Mann-Whitney test, p=0.0080 and 0.0426, respectively). Conversely, the frequency of the IL-2 +CD8+ phenotype in IND was lower in the absence of SAg than in the presence of SAg (IND and IND*, Wilcoxon test, p=0.0078). Furthermore, in the presence of cellular stimulation by SAg, the proportion of IFN-g+CD8+ cells was lower in IND than in TDC (Mann-Whitney test, p=0.0464). Similarly, we observed that the frequency of this phenotype was significantly lower in all chagasic individuals, even in those

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Cytokines in Chagas disease and HIV infection Tozetto-Mendoza TR et al.

to induce a suppressive immune response like the response observed in this study using trypomastigote antigen (20). Third, despite the limited sample size, we observed a polarized Th1 CD8+ T cell response when cells from chagasic individuals were exposed in vitro to SAg stimuli, thus distinguishing the typical digestive/cardiac form (TDC group) from the indeterminate form of CD (IND group) (Figure 4). In the TDC group, we clearly verified an up-regulation of inflammatory cytokines on CD8+ T cells challenged with soluble trypomastigote antigen. However, the biased inflammatory response characterizing individuals with the cardiac and/or digestive form of CD (33) could be justified by the absence of the activation of a regulatory immune mechanism. Moreover, the profile of cytokines observed in the IND subgroup has been previously suggested to be a strategy that improves the chances of parasite survival (28). Additionally, CD8+ T cells seem to play a role in the balance between proinflammatory responses to eradicate the pathogen and antiinflammatory responses to limit inflammation as described in CD and other infections (34,35). In fact, the TDC sub-group presented a lower frequency of IL-10+CD8+ cells and a higher frequency of IFN-g+CD8+ cells than individuals with the indeterminate form of CD (IND). This was especially true for individuals with HIV infection (Figure 4, red points). Nevertheless, studies on the source of IL-10 in humans are conflicting, partly due to the different assessment methods used (36,37,38). Thus, we also assessed the production of IL-10 by monocytes with and without SAg stimulation, but we did not find any significant difference between the clinical groups (data not shown). On the other hand, we were able to detect a significant difference between the IND and TDC sub-groups in relation to the IL-10+CD8+ phenotype. Our findings corroborate the fact that IL-10 produced by T cells seems to act mainly as a regulatory cytokine to protect the infected hosts against unwanted excessive Th1 activation and severe inflammatory pathology (20,29,34). Our patients exhibited a trend of higher IL-4 production by SAg-stimulated CD4+ T cells in the co-infected (CO) group compared with the HIV group, similar to the patients studied by Rodrigues et al. (2005) (18). The percentage of IL-4-producing CD4+ T cells stimulated by SAg varied from zero to 27.47% with a median of 10.71% in the CO group, while in HIV group, the same cell population was between zero and 46.79% with a median of 0.0% (p=0.0957). Moreover, the same tendency was noted by comparing the CO and CHR groups, as the former tended to show higher percentages of SAg-stimulated IL-4-producing CD4+ T cells than the latter. The percentage of IL-4-producing SAgstimulated CD4+ T cells varied from zero to 27.47% with a median of 10.71% in the CO group, while that of the CHR group varied from zero to 20.84% with a median of 4.24% (p=0.1108). This trend was not observed when we compared the non-SAg-stimulated CD4+ T cells of the CO group with those of the HIV or CHR groups. These data suggest that SAg can induce the synthesis of IL-4 and that the co-infected group is more sensitive to this stimulus. Recently, studies have characterized the adaptive immune response in different epidemiologic contexts. In fact, the interference of previous exposure to a specific antigen (vaccinated or not) has been reported in both dual infections or other models of infections involving HIV. In the infection model of Schistosoma in mice, the suppression of the immune response to a Th1-type HIV vaccine and impaired expansion of pathogen-specific cytotoxic CD8+ T cell responses was

Figure 4 - Proportion of cytokine-producing CD8+ T cells in chagasic sub-groups. (A) IL-2+CD8+. (B) IFN-c+CD8+. (C) IL-10+ CD8+. Patients were subdivided into a typical digestive and/or cardiac manifestation (TDC) sub-group and a non-cardiac/nondigestive manifestation of CD (IND) sub-group, and all chagasic (TDC and IND) patients were identified as the ALL group. The asterisk (*) represents the condition in which cells were stimulated with soluble T. cruzi antigen. The TDC and IND subgroups had four HIV-infected individuals each. Red plots indicate HIV-infected individuals. The Mann-Whitney test was used for between-group comparisons (p indicated below the x-axis), and the Wilcoxon test was used for within-group comparisons (p indicated above the x-axis). The difference between TDC* and IND* stimulated T cells to produce cytokines was significant for the IFN-g+CD8+ phenotype (Mann-Whitney test p=0.0464). The potential capability of T cells to produce cytokines was different between TDC and IND for the IL-2+CD8+ phenotype (Mann-Whitney test, p=0.0080) and the IL-10+CD8+ phenotype (Mann-Whitney test, p=0.0426). No significant differences were found in relation to the other phenotypes.

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reported (21). In addition, in children living with HIV, higher CD4+ T cell activation leads to poor vaccine response, which may be related to a disequilibrium of T regulatory responses (39, 40). In infant macaques vaccinated with BCG or M. tuberculosis, CD4+ T cells were persistently activated in oral and/or gastrointestinal tissues, which may have facilitated oral SIV infection, according to the authors (41). Moreover, in HIV-infected Kenyan infants, higher central memory Th1 responses to M. tuberculosis antigens were observed at three months, but reduced effector memory Th1 responses to vaccine antigens were seen at three and 12 months. Long-term monitoring of vaccine efficacy and T-cell immunity in this vulnerable population is warranted (42). Our study, however, has some important limitations. First, because it was not possible to obtain serial CD4+ cell counts, HIV viral loads or parasitological data, it would be very useful to verify additional relationships with immune responses. This fact is directly linked to the limited access patients with CD have to medical assistance and diagnosis. Second, our critical findings were based on a limited number of available samples. However, we screened a rare and wellcharacterized group of HIV and/or chagasic individuals.

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’ ACKNOWLEDGMENTS

16.

The authors would like to thank Mrs. Erika Gakiya from the Laboratory of Parasitology (LIM46) of HCFMUSP and Mrs. Célia Regina Furucho from the Laboratory of Immunology (LIM 48) of HCFMUSP for technical support and to Prof. Ises A. Abrahamson from the Department of Immunology of the Institute of Biomedical Sciences of the University of São Paulo for supplying soluble T. cruzi trypomastigote antigens. Sponsorship: Japan International Cooperation (JICA) and State of São Paulo Foundation for Research Support (FAPESP 2012/50273-0).

17.

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’ AUTHOR CONTRIBUTIONS

19.

Tozetto-Mendoza TR and Vasconcelos DM were responsible for writing and revising the manuscript, performing the cellular assays, analysing the data and supervising the study. Ibrahim KY and Sartori AM were responsible for the clinical analysis of the participants and paper revision. Bezerra RC and de Freitas VL were responsible for performing the parasitological tests. Shikanai-Yasuda MA was responsible for the study coordination and for writing and critically revising the manuscript.

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CLINICAL SCIENCE

Assessment of the quality of life, muscle strength, and dynamic balance of elderly Kendo players Da´rio Lucas Costa de Mendonça,I,II,* Angelica Castilho Alonso,I,II,III Ju´lia Maria D’Andrea Greve,I Luiz Eugeˆnio Garcez-LemeI,II I Laboratorio de Estudo do Movimento, Laborato´rio de Investigaçaõ Me´dica do Sistema Mu´sculo-Esquele´tico LIM 41, Instituto de Ortopedia e Traumatologia, Hospital das Clıńicas (HCFMUSP), Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. II Grupo de Ortogeriatria, Instituto de Ortopedia e Traumatologia, Hospital das Clıńicas (HCFMUSP), Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. III Programa de Mestrado em Ciencias do Envelhecimento, Universidade Sao Judas Tadeu, Sao Paulo, SP, BR.

OBJECTIVE: To compare Kendo players with active elderly adults in terms of quality of life, functional aspects (muscle strength, postural balance) and body composition. METHODS: This was a controlled cross-sectional study. Twenty elderly individuals were divided into two groups: the Kendo group, with an average age of 71.8 (5.4) years, and the Control group, with an average age of 73.1 (4.8) years. Quality of life was evaluated using a questionnaire (WHOQOL-bref and WHOQOL-old); body composition was evaluated with a bioimpedance scale (InBody230); hand-grip strength was assessed with a portable manual dynamometer (Jamar SH 5001); flexor and extensor muscle strength of the knees was evaluated with an isokinetic device (Biodexs System 3 model Biodex Multi Joint System, BIODEX); and dynamic balance was assessed using a force platform (Balance Master System, Neurocom International, Inc.,s Clackamas County, Oregon, USA). RESULTS: The groups were statistically homogeneous in terms of socio-demographic characterization, body composition, muscle strength, and dynamic balance, but the Control group was faster in the sit-to-stand test (p=0.03). The Kendo group had a statistically significantly better quality of life; in the WHOQOL-bref, these differences were present in the physical (pp0.001) and environment (p=0.004) domains, and in the WHOQOL-old, these differences were present in social participation (p=0.001) and in past, present, and future activities (p=0.019). CONCLUSION: The results suggest that Kendo is a health-promoting activity that improves the quality of life, functional aspects (muscle strength and postural balance) and body composition of players. KEYWORDS: Aged; Martial Arts; Muscle Strength; Postural Balance; Quality of Life. de Mendonc¸a DL, Alonso AC, Greve JM, Garcez-Leme LE. Assessment of the quality of life, muscle strength, and dynamic balance of elderly Kendo players. Clinics. 2017;72(11):661-666 Received for publication on February 13, 2017; First review completed on April 28, 2017; Accepted for publication on July 20, 2017 *Corresponding author. E-mail: mendoncadlc@gmail.com

’ INTRODUCTION

QoL has been defined by the World Health Organization as "the individuals’ perception of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards, and concerns" (3). To improve the QoL and life expectancy of the elderly, regular physical activity is recommended (4). Kendo is a martial art that has its origin in kenjutsu (samurai fencing) and seeks to shape the mind, body, and spirit through correct training in sword handling (5). The cultural and social aspects of Kendo have been studied in depth, but its impact on health and QoL is unknown. To promote adherence to physical activity programs, such activities should accommodate the participant’s lifestyle, have cultural context and respect cultural traditions to improve the participant’s values and meet his or her needs (6). For this reason, the objective of this study was to compare Kendo players with active elderly adults in terms of QoL, functional aspects (muscle strength and postural balance) and body composition.

With increased life expectancy, there is a growing interest in the study of quality of life (QoL) with the aim of recognizing healthy populations and highlighting their healthpromoting practices. Therefore, it is important to identify cultural groups to study the strategies they have adopted and to disclose different ways of ageing well (1). From this perspective, the improved QoL of the population must be considered a primary objective of our activities as scientists and clinicians (2).

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’ MATERIALS AND METHODS

aqua aerobics twice a week. Two participants (20%) practiced yoga once a week. Two participants (20%) practiced Tai Chi Chuan once a week.

Study Type and Ethics This was a controlled cross-sectional study conducted at the Motion Study Laboratory of the Institute of Orthopaedics and Traumatology, Clinical Hospital, University of São Paulo School of Medicine. It was approved by the Ethics Committee of the School of Medicine of the University of Sao Paulo (Universidade de São Paulo- USP) under N. 012/14.

Procedures The elderly players were invited to participate in the Kendo group during Kendo events at Sao Paulo State. The elderly physically active individuals were invited to participate in the Control group at the University for the Third Age of the University of Sao Paulo (Universidade de São Paulo– USP). The groups were paired by age, and all subjects signed an Informed Consent Form.

Description of the Sample A convenience sample of twenty elderly adults divided into two groups paired by age were assessed: the Kendo group, comprising 10 players, and the Control group, comprising 10 physically active elderly adults that did not practice Kendo. All the participants underwent a QoL evaluation. For the functional evaluation, two players did not attend because they lived far away from the laboratory, but this fact did not interfere with the primary outcomes of the study. The inclusion criteria for both groups were as follows: male volunteers aged X60 years who were autonomous and had a positive self-perception of their health, regardless of comorbidities; absence of vestibular, proprioceptive, auditory, or neurological impairment and/or any mental disturbances or disorders; no use of medications that might compromise postural balance or that interfere with musculoskeletal metabolism; absence of lesions, surgery, or disease over the previous six months that might cause lower-limb joint limitations; absence of any lower-limb dysmetria; presence of clinically normal gait without claudication. For the Kendo group, more than five years of practicing the martial art was required. For the Control group, participants were required to have no involvement with Kendo and to participate regularly in physical activities. The exclusion criteria were an inability to understand and respond to the applied questionnaires or a request to leave the study. Physical activity level was classified according to the brief IPAQ (7). In the Kendo group, five participants were classified as very active (50%) and five as active (50%); in the Control group, four participants were classified as very active (40%) and six as active (60%). In the Kendo group, eight participants (80%) practiced twice a week, one participant (10%) practiced three times a week, and one participant (10%) practiced five times a week. Iaidô (withdrawal-of-sword technique) was practiced by four participants (40%) once a week. All the Kendo players had practiced for more than five years and were ranked at the Dan level: one player (10%) was ranked as 1st Dan, one (10%) as 2nd Dan, three (30%) as 4th Dan, and five (50%) as 7th Dan. Two participants (20%) exercised with a resistive load twice a week. One participant (10%) participated in aqua aerobics twice a week. One participant (10%) engaged in vigorous walking five times a week. In the Control group, eight participants (80%) performed vigorous walking, as follows: three participants (30%) walked seven times a week, three (30%) walked four times a week, and one (10%) walked twice a week and ran once a week. Eight participants (80%) exercised with a resistive load: three participants (30%) did so once a week, three (30%) did so twice a week, one (10%) did so three times a week, and one (10%) did so five times a week. One participant (10%) participated in

Quality of Life Assessment The QoL assessment was conducted with the WHOQOLbref and WHOQOL-old questionnaires. The participants selfadministered the questionnaires to avoid embarrassment and influencing responses (8). The WHOQOL scores were calculated on a computer using Excel (9).

Anthropometric Assessment and Body Composition Height in centimetres was measured with a stadiometer. For this measurement, the distance between the platform of the stadiometer and the vertex of the head was considered, using the Frankfurt plane as the basis. Body mass in kilograms, skeletal muscle mass, and body mass index (BMI) were measured using an InBody230 bioimpedance scale.

Muscle Strength Assessment Two assessment protocols were used. 1) Hand-grip strength: A manual Jamars dynamometer was used with a measurement adopted by the American Society of Hand Therapists (ASHT). The hands were switched after each manoeuvre, with one minute of rest between tests; the first manoeuvre was performed with the dominant limb. The first two manoeuvres were performed to familiarize the participant with the test, and then three additional manoeuvres were performed with each hand. For analysis purposes, the average of the last three measurements for each limb was calculated (10). 2) Knee extension and flexion strength: A Biodexs multijoint System3 isokinetic dynamometer (Biodex Medical Systems, Inc., Shirley, NY, USA) was used. Concentric movements were chosen (knee flexion and extension) starting at 90o of flexion and proceeding to 20o of extension and were corrected by the gravitational force at half amplitude according to the manufacturer’s instructions. The angular velocity of choice was 60o/second because it allows the recruitment of a greater number of motor units for rapid muscle contraction strength. To familiarize the participants with the device, four submaximal repetitions were performed, and each was followed by an interval of 60 seconds. For the test, two series of five repetitions without interruption were performed for knee extension and flexion. Between the series, there was an interval of 60 seconds. The test started with the dominant limb, and after 60 seconds, the test was performed with the non-dominant limb. For data analysis, the values of the second series of each measured limb were used, taking into consideration the effects of motor familiarity related to repetition (11).

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’ RESULTS

Assessment of Dynamic Balance. Each assessment was repeated three times with an interval of 30 seconds between repetitions. Two assessment protocols were used: 1) Sit-to-stand test: The participant was seated on a bench without a backrest or arm support with the knees at 90o flexion and the feet 10 cm apart at the heels. Upon a visual signal (green light) and a verbal command (‘‘stand up’’), the participant stood up from the bench and remained motionless, looking at a fixed point at eye height, with the arms relaxed beside the body (12). 2) Stair-climbing test: The participant was placed in an orthostatic position in front of a step that measured 20 cm high. Upon a visual signal (green light) and a verbal command (‘‘climb up’’), the participant climbed up the step with the left foot and climbed down the step with the right foot without supporting the right foot on the step; then, the participant climbed down with the left foot parallel to the right foot. After climbing down the step, the participant remained motionless, looking at a fixed point at eye height, with the arms relaxed beside the body. At the end of three measurements, the order of the feet was reversed so that the participant climbed up with the right foot and down with the left foot.

Subject Characteristics The groups presented no statistically significant differences (Table 1).

Quality of Life Assessment The Kendo group showed a statistically significantly higher QoL; on the WHOQOL-bref, these differences were evident in the physical and environment domains, and on the WHOQOL-old, these differences were evident in social participation and in past, present, and future activities (Table 2).

Functional assessment (muscle strength and dynamic balance) The groups presented no statistically significant differences in BMI, height, body mass, muscle mass, hand-grip strength, and knee extension and flexion force (Table 3). The Control group was faster in the sit-to-stand test (Table 4).

’ DISCUSSION The martial arts are physical exercises in which each style has its specific benefits, and their contributions to health have been demonstrated in several studies (5,13). However, there is a lack of academic publications regarding the benefits of Kendo. When searching PubMed for the terms ‘‘Kendo’’, ‘‘elderly’’, and ‘‘quality of life’’, no reference to the topic of interest was found. We assessed a convenience sample of 20 participants and considered the results as a possible pilot for future studies. The main findings of this study were that the practice of Kendo improves the QoL of elderly players, allowing them to remain physically active and maintain good functionality.

Statistical Analysis The data were stored and analysed in SPSS 20.0 for Windows and are presented as averages, medians, and standard deviations. The Mann-Whitney U test was used to compare the quantitative data, and the chi-square test was used to compare qualitative data. A 5% significance level was adopted throughout the statistical analysis.

Table 1 - Socio-demographic characteristics and comorbidities of the Kendo and Control groups. Kendo N (%)

Control N (%)

w2 (p)

White Black Yellow

1 (10%) 0 (0%) 9 (90%)

2 (20%) 1 (10%) 7 (70%)

1.58 (0.453)

Married Widow

9 (90%) 1 (10%)

10 (100%) 0 (0%)

9 to 11 years 12 to 16 years 417 years

2 (20%) 2 (20%) 6 (60%)

0 (0%) 4 (40%) 6 (60%)

Yes No

8 (80%) 2 (20%)

10 (100%) 0 (0%)

Race or Colour

Marital Status 1.05 (0.305)

Education Level 2.66 (0.264)

Retired 2.22 (0.136)

Family Income o1 minimum wages 1 to 2 minimum wages 2 to 3 minimum wages 3 to 5 minimum wages 5 to 10 minimum wages 10 to 20 minimum wages More than 20 minimum wages Comorbidities Systemic arterial hypertension Diabetes mellitus Orthopaedic diseases History of previous fractures History of previous falls (p12 months)

1 2 0 2 1 2 2

(10%) (20%) (0%) (20%) (10%) (20%) (10%)

0 1 3 2 3 1 0

(0%) (10%) (30%) (20%) (30%) (10%) (0%)

6.44 (0.168)

4 0 2 6 1

(40%) (0%) (20%) (60%) (10%)

6 2 0 2 3

(60%) (20%) (0%) (20%) (30%)

0.80 2.22 2.22 3.33 1.25

Chi-square. *pp0.05.

663

(0.37) (0.13) (0.13) (0.68) (0.26)

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CLINICS 2017;72(11):661-666

Table 2 - Comparison of age, quality of life, WHOQOL-bref domains and facets of the WHOQOL-old between the Kendo and Control groups. Kendo Median

Kendo Average (sd)

Control Median

Control Average (sd)

Age (years) Quality of life

70 4.30

71.8 (5.4) 4.39 (0.32)

73.50 3.72

73.10 (4.8) 3.76 (0.30)

0.38 0.002*

Physical Psychological Social relations Environment

4.21 4.17 4.17 4.25

4.39 4.25 4.17 4.23

(0.32) (0.36) (0.53) (0.36)

3.79 4.08 3.83 3.50

3.64 3.85 3.80 3.53

(0.34) (0.59) (0.55) (0.54)

p0.0001* 0.218 0.143 0.004*

Sensory abilities Autonomy Past, present, and future activities Social participation Death and dying Intimacy

4.50 4.25 4.38 4.50 4.25 4.63

4.30 4.00 4.40 4.43 4.15 4.45

(0.64) (0.80) (0.47) (0.35) (0.86) (0.55)

4.25 3.75 3.88 3.75 3.50 4.13

3.95 3.45 3.88 3.75 3.75 4.03

(0.71) (0.71) (0.32) (0.31) (0.91) (0.51)

0.190 0.063 0.019* 0.001* 0.280 0.089

WHOQOL-bref

WHOQOL-old

Mann-Whitney U. *pp0.05. Legend: sd-standard deviation.

Table 3 - Comparison of body mass index, height, body mass, muscle mass, hand-grip strength, and knee extension and flexion force between the Kendo and Control groups. Kendo Median 2

Body Mass Index (kg/m ) Height (cm) Body mass (kg) Muscle mass (kg) Hand-Grip Strength Dominant (kg) Non-dominant (kg) Knee Extension TP/BW dominant leg (%) TP/BW non-dominant leg (%) TW dominant leg (J) TW non-dominant leg (J) Knee Flexion TP/BW dominant leg (%) TP/BW non-dominant leg (%) TW dominant leg (J) TW non-dominant leg (J)

25.20 165.50 68.20 28.55 39.67 38.83

Kendo Average (sd) 24.8 164.5 67.2 28.1

Control Median

(3.3) (5.0) (9.0) (2.9)

Control Average (sd)

25.65 167.50 64.30 27.00

40.50 (5.65) 39.17 (4.04)

25.34 166.6 70.70 28.84

40.99 38.00

(3.8) (8.8) (15) (5.7)

p 0.83 0.57 0.76 0.83

39.99 (6.61) 37.43 (5.74)

0.86 0.48

181.20 179.70 475.20 458.55

175.59 182.53 449.75 483.29

(44.91) (38.82) (112.92) (92.51)

186.05 179.10 471.35 483.75

179.98 174.96 483.14 489.02

(31.05) (26.01) (114.91) (123.41)

0.72 0.79 0.66 0.93

105.45 103.55 324.85 305.60

103.8 99.03 310.95 305.68

(26.99) (16.19) (81.38) (59.80)

91.95 89.20 277.9 306.35

94.74 95.73 289.38 307.00

(20.35) (22.73) (77.37) (74.70)

0.48 0.59 0.66 1.00

Mann-Whitney U *pp0.05. Legend: cm – centimetres; kg – kilograms; TP/BW – torque peak adjusted for body weight; TW – total work; N-m – Newton-metres; % – percentage; J – Joules.

Table 4 - Comparison of balance on the sit-to-stand and stair climbing tests between the Kendo and Control groups. Kendo Median

Kendo Average (sd)

Control Median

Control Average (sd)

Transfer time (seconds) Body mass transfer to stand up (%) Balance velocity (˚/second)

0.61 19.50 2.8

0.60 (0.17) 29.38 (30.34) 2.74 (0.77)

0.39 20.00 3.6

0.40 (0.16) 21.80 (6.27) 3.96 (1.12)

0.03* 0.72 0.06

Body mass transfer to climb up DS (%) Body mass transfer to climb up NDS (%) Difference between limbs for climbing up (%) Movement time DS (seconds) Movement time NDS (seconds) Time difference between limbs (%) Body mass transfer to climb down LND (%) Body mass transfer to climb down DS (%) Impact difference between limbs while climbing down (%)

38.50 34.50 8.08 1.78 1.65 8.06 39.00 45.50 17.39

37.88 35.13 8.18 1.76 1.68 6.51 36.63 42.13 15.05

41.00 39.00 2.19 1.64 1.69 -0.62 43.50 45.00 4.09

41.50 37.80 19.59 1.71 1.77 -1.02 45.30 49.90 10.20

0.53 0.56 0.79 0.37 0.72 0.29 0.27 0.79 0.48

Sit-to-Stand

Stair Climb

Mann-Whitney U *pp0.05. Legend: DS – dominant side; NDS – non-dominant side; sd – standard deviation.

664

(9.64) (8.36) (14.94) (0.29) (0.35) (11.94) (11.01) (12.60) (12.66)

(9.83) (13.14) (41.65) (0.25) (0.39) (13.73) (18.83) (25.69) (37.80)

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group was more vigorous than that performed by the Kendo group because according to Pau et al. (22) and Brech et al. (23), vigorous physical activity yields more efficiency when transferring from sitting to standing. The clinical implication of this study is that those who practice Kendo while ageing improve their QoL. However, the sample size of the two groups is a limitation of the study; therefore, despite being sufficient in terms of statistics (demonstrating a good internal validity), the results must be analysed with caution when applied to the broader population. Future studies with more participants will be performed to identify and describe the benefits of physical activity for the QoL of the elderly.

The "physical domain" score was higher for the Kendo group and, according to Molzahn et al. (14), is the most important aspect of the assessment. Sampaio and Ito (15) assessed 465 elderly individuals in Japan, and physical activity was demonstrated to have a great influence on the WHOQOL-bref measurement scores. For the WHOQOL (3), physical self-perception depends on how satisfied an individual is with his/her physical and functional condition regardless of the number of comorbidities. Reinforcing this information, the present study demonstrates that superior physical functionality is not enough to guarantee a better QoL because although the Control group scored significantly higher on the functional assessment, the Kendo group scored significantly higher on the WHOQOL evaluation, especially in the physical domain. The "environment domain" score was higher for the Kendo group. According to Fleck et al. (16), this domain is related to participation and leisure opportunities and to opportunities to acquire new information and skills. Sonati et al. (17) assessed the QoL of 15 kenjutsu players in the city of São Paulo (12 men and three women with an average age of 27.73±7.40 years) and concluded that the physical domain positively influences the psychological domain, which in turn directly influences the environment domain. Pereira et al. (18) and Vagetti et al. (19) reported that the physical domain directly influences the environment domain because an elderly individual with a higher physical self-perception will take more advantage of leisure opportunities and will have better control of his or her environment. This information corroborates the results of the present study because the Kendo group demonstrated statistically significant superiority in the physical domain and, consequently, in the environment domain. The "past, present, and future activities" facet scores were higher for the Kendo group. According to Power et al. (8), this facet evaluates satisfaction regarding life achievements. This result corroborates the tenets of the Japanese Kendo Federation (5), which advocates "Kendo for life". The "social participation" facet score was higher for the Kendo group. According to Power et al. (8), this facet concerns not both activities in the community and everyday activities. This result corroborates the tenets of the Japanese Kendo Federation (5), which posits that contributing to the peace and prosperity of all and serving the country and the society with love are among the values that should be practiced. We found no statistically significant difference in sociodemographic characteristics, comorbidities, BMI, height, body mass, muscle mass, hand-grip strength, and knee flexor and extensor strength. Tanimoto et al. (20) believe that there is an association between strength and functional capacity when assessing muscle mass, hand-grip strength, and physical performance. Alonso et al. (21) also reported that hand-grip strength correlates with knee flexor and extensor strength to positively affect dynamic balance. Therefore, because there was no significant difference in the strength and muscle mass results, it was expected that there would be no difference in dynamic balance, similar to the findings of Serra et al. (6). However, in our study, when we compared balance in the sitto-stand test, the Kendo players took longer to perform weight transfer. This difference was significant, but no clinical implications related to balance were observed because the groups were similar in terms of history of falls. This result suggests that the physical activity performed by the Control

’ ACKNOWLEDGMENTS This article is the result of a dissertation for obtaining a Master of Sciences degree from the Orthopaedics and Traumatology Program of the School of Medicine of the University of Sao Paulo.

’ AUTHOR CONTRIBUTIONS de Mendonc¸a DL was responsible for the study design, data collection, data analysis and preparation of the manuscript. Alonso AC was responsible for data collection, data analysis and critical review of the manuscript. Greve JM was responsible for the critical review of the manuscript. Garcez-Leme LE supervised the study and was responsible for the study design and critical review of the manuscript.

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CLINICAL SCIENCE

Cardiorespiratory performance of coronary artery disease patients on land versus underwater treadmill tests: a comparative study Mauricio Koprowski Garcia,I,* Limanara Rizzo,II Paulo Yazbek-Ju´nior,I Daniela Yutiyama,I Fabiola Jomar da Silva,I Denise Matheus,I Luiz Eduardo Mastrocolla,III Eduardo MassadII I

Instituto de Medicina e Reabilitacao, Hospital das Clinicas (HCFMUSP), Faculdade de Medicina, Universidade de Sao Paulo, Sao Paulo, SP, BR. Departamento de Medicina Legal e Etica Medica, Faculdade de Medicina (FMUSP), Universidade de Sao Paulo, Sao Paulo, SP, BR. III Instituto de Cardiologia Dante Pazzanase, Sao Paulo, SP, BR. II

OBJECTIVE: To compare responses to a cardiopulmonary exercise test on land versus on an underwater treadmill, to assess the cardiorespiratory performance of coronary artery disease patients while immersed in warm water and to compare with the performance of healthy individuals. METHODS: The sample population consisted of 40 subjects, which included 20 coronary artery disease patients aged 63.7±8.89 years old, functional class I and II, according to the New York Hearth Association, and 20 healthy subjects aged 64.7±7.09 years old. The statistical significances were calculated through an ANOVA test with a (1 - b) power of 0.861. ClinicalTrials.gov: NCT00989248 (22). RESULTS: Significant differences were uncovered in coronary artery disease group regarding the variables heart beats (HB), (p40.01), oxygen consumption (VO2), (p40.01) and carbon dioxide production (VCO2) (po0.01). Also, for the same group, in relation to the environment, water versus on land for HB, VO2, VCO2 and oxygen for each heart beat (VO2/HB) all of than (po0.01). The stages for data collected featured the subject’s performance throughout the experiment, and within the given context, variables rating of perceived exertion (RPE), HB, VO2, VCO2 and VO2/HB (po0.01) showed significant interactions between test stages and environment. Additionally, there was a significant interaction between the etiology and the test stages for the variables HB, VO2 and VCO2 (po0.01). Electrocardiographic changes compatible with myocardial ischemia or arrhythmia were not observed. The subjects exhibited lower scores on Borg’s perceived exertion scale in the water than at every one of the test stages on land (po0.01). CONCLUSION: This study show that a cardiopulmonary exercise test can be safely conducted in subjects in immersion and that the procedures, resources and equipment used yielded replicable and reliable data. Significant differences observed in water versus on land allow us to conclude that coronary artery disease patients are able to do physical exercise in water and that the physiological effects of immersion do not present any risk for such patients, as exercise was well tolerated by all subjects. KEYWORDS: Cardiopulmonary Stress Test; Immersion; Individual Capability; Exercise. Garcia MK, Rizzo L, Yazbek-Ju´nior P, Yutiyama D, Silva FJ, Matheus D, et al. Cardiorespiratory performance of coronary artery disease patients on land versus underwater treadmill tests: a comparative study. Clinics. 2017;72(11):667-674 Received for publication on February 20, 2017; First review completed on May 30, 2017; Accepted for publication on July 21, 2017 *Corresponding author. E-mail: mauriciokg@usp.br

’ INTRODUCTION

conditioning (1). Cardiopulmonary exercise tests (CPX) conducted on land are well known. When conducted in water, however, they require additional care as well as suitable equipment and resources to comply with the provisions set forth by the ‘‘clinician’s guide to CPX in adults: as cientific statement from the American Heart Association, 2010’’ (2). During immersion at the level of the manubrium, there is compression on surface veins caused by hydrostatic pressure. Blood flow in lower limbs is reversed from downward to upward through the unidirectional valves, first to the thighs and then upward to the abdomen and finally to the chest and to the heart. During immersion up to the iliac crest, changes in volume are not significant, but central venous pressure begins to increase during immersion at the level of

Physical activity in a warm water pool is traditionally used in rehabilitation facilities with patients who have different pathologies. As exercise in water can be reasonably easy to perform, it may enhance mobility, strength and physical

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Thus, the aim of this study was to compare responses to a cardiopulmonary exercise test (CPX) on land versus on an underwater treadmill, to assess the cardiorespiratory performance of coronary artery disease (CAD) patients while immersed in warm water and to compare with the performance of healthy (H) individuals.

the manubrium and eventually gets higher during total immersion (3). Arborelius et al., 1972 (4), showed that during immersion at the neck level, an increase in central blood volume of 700 ml is observed, which represents a 60% increase in the central volume. One-fourth of the volume, that is 180-240 ml, is oriented to the heart, thus causing the four heart chambers to dilate (5-7). The heart volume increases by 27-30% during immersion at neck level (8). The heart, however, is not a static receptacle and its physiological response to increased volume is a rise in the force of contraction the greater the myocardial distension, the greater (more efficient) the muscle contraction, according to the FrankStarling law (9). Most cardiovascular changes depend on water temperature and level of immersion. A progressive increase in cardiac output was observed at higher temperatures, which comprised an increase by 30% at 33oC to an increase by 121% at 39oC (10). During immersion at the neck level, systemic vascular resistance decreases by 30% as a result of reduced sympathetic vasoconstriction, and it remains low for a few hours following the first hour of immersion. This effect is also temperature dependent, as the temperature increases, the magnitude of the reduction becomes greater (3,5). Lung blood flow also increases as a result of increased central volume and blood pressure. Average lung artery pressure ranges between 5 mmHg on land and 22 mmHg during immersion at the neck level. Most of the lung blood volume is directed to the larger vessels of the lung vascular bed (6). Immersion is also associated with renal responses such as increased diuresis, decreased plasma concentration, natriuresis, kaliuresis and suppression of arginine vasopressin, plasma rennin and aldosterone, with consequent augmentation of renal free water excretion (3,9). Several studies have assessed and validated the use of aquatic therapy for cardiovascular rehabilitation following heart failure and ischemic heart disease. Common sense clinical opinion is that immersion in warm water and controlled exercises are well tolerated by patients diagnosed with coronary artery disease (CAD), even when they experience the physiological effects discussed herein. The assumption is that there are no major differences between a CPX conducted in water and its counterpart on land. Additionally, the required equipment and resources are supposed to be similar, provided that there is compliance with the required test preparation methodology for the CPX conducted in water.

’ METHODS The sampling population of this study comprised 40 subjects, 20 of whom were healthy controls and 20 of whom were patients enrolled in the physical conditioning program of the Institute of Medicine and Rehabilitation, Clinics Hospital, School of Medicine, University of São Paulo. The patient group had a medical history of one or more of the following conditions: acute coronary syndrome, coronary angioplasty, revascularization surgery or CAD. Patients were between 55 and 80 years old, and they were first examined by a heart specialist who assessed their clinical history and validated their medical conditions. Any patient who did not comply with the test schedule or those who had peripheral artery disease, diabetes, chronic lung disease, hypertension (blood pressure 4160/90), or significant or unstable cardiovascular morbidity conditionswhile taking medication were excluded from the study (Table 1). ClinicalTrials.gov: NCT 00989248.

Test Design Both groups underwent two CPX tests and were always monitored by the same cardiology specialist throughout the testing and that did the data interpretation. All of the participants received clear explanations about the test methodology and granted their consent, after which they were requested to sign an informed consent. Clinical data were then collected, and body mass and height were measured. Within an interval of 3-7 days after the land CPX was conducted, the second CPX was conducted in a warm indoor pool with 48 m3 of water volume and depths ranging between 1.10 and 2.10 m. The nursing staff prepared the subjects for both tests by first cleaning the skin areas where electrodes were applied, connecting the cables, putting the facemasks on the subjects and finally checking their systolic and diastolic blood pressure (SDBP). The gas analyzer was calibrate prior to every run of the test.

Table 1 - Baseline characteristics of subjects. CAD N Males Age (years) Weight (Kg) Height (cm) Body Mass Index - BMI (kg/m?) Patients Under Medication Antihypertensive Diuretic Hypercholesterolemia Functional class I VO2 max 420ml/kg-1 min-1 Functional class I VO2 (AT)414ml/kg-1 min-1 Functional class II VO2 max from 16 to 20mL/kg-1 min-1 Functional class II VO2 (AT) from 11 to 14mL/kg-1 min-1 Base Etiology - Revascularization/Stent

20 20 63.7 73.7 168 22.83

Healthy

±8.89 ±10.6 ±5.95

20 20 64.7 73.6 170.6 23.19

±7.09 ±18.8 ±11.7

18 6 9 8 8 12 12 12/8

Coronary Artery Disease (CAD); Standard Deviation (SD); Number of participants (n); Functional Class as per the New York Heart Association (NYHA). Maximum rate of oxygen consumption (VO2 max); Oxygen volume (VO2); Anaerobic Threshold (AT).

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On land, the environment temperature was kept stable at 20±2oC and the relative air humidity was maintained at 50-70% (2). In the pool, the water temperature was maintained at 33-34oC and the relative air humidity between 60 to 80%. Subject test preparation for the water test followed the same procedures as for the on land test. To improve the conduction of electrical impulses without causing any signal interference, however, the electrocardiogram electrodes received additional protection by applying a 3M occlusive bandage on the top of each of the cable terminals. The subject’s adaptation to the water was check by a pool specific practical test in order to identify and exclude from the study those individuals who showed difficult adaption to or fear of the water. A brief simulation allowed subjects to become acquainted with the environment and the equipment, thus lowering their level of anxiety. Due to a lack of interfaces connected directly the underwater treadmill to a computer, the Bruce Protocol (20) was used to be the most suitable. Perceived levels of exertion and/or dyspnea were assessed as per the Borg rating of perceived exertion (21). A set comprising a facemask covering the nose and mouth, a cap and a flow sensor was used to conduct the air exhaled by subjects to the gas analyzer. An model Metalyzer II (Cortex CPET) measured respiratory and metabolic variables. As this device was connected to the Ergo-Elite software on the computer, it supplied the following data: exhaled gas (average min), oxygen consumption (VO2), carbon dioxide production (VCO2), oxygen equivalents (VE/VO2) and carbonic dioxide equivalents (VE/VCO2) as well as the respiratory exchange rate (RER - VCO2/VO2). Meditrace electrodes connected to a special cable with five thoracic derivations sent electrical signals to the digital Micromed ECG, thus allowing for continuous ECG monitoring by the researcher. A Model MF621 sphygmomanometer was placed on each subject’s right arm to check SDBP. During the water test, due to the unfavorable access conditions, SDBP was checked at the beginning of the test and two minutes after the test was completed. For the land test, the KT-ATL Millenium treadmill by Inbramed/ Inbrasorpts was used and for the water test, the

Aquafit Hi-Tecs by Sahinco. The latter, made in stainless steel and featuring an electric-hydraulic unit, prevented accidental electric shock. Produced speeds ranging from 2 up to 10 km/h at 0-13% inclines, same inclination as the land treadmill, through electronic programming without a computer interface (Figure 1). The test started at stage 1 of the Bruce Protocol, with 3 minutes of activity at a 10% incline and a speed of 2.7 km/ h, and was completed when the Respiratory Quotient (RQ) reached X1.1. After the test was completed, subjects kept walking on the treadmill for 2 minutes at a speed of 2.7 km per hour and 0% incline while still being monitored. The test was interrupted whenever any of the criteria set by the Brazilian Cardiology Society/American Heart Association were not complied with (12). Data were collected at 5 relevant test stages or cardiorespiratory levels: One, rest (REP), accelerated ventilation, heart beat and oxygen consumption were observed to indicate whether subjects were anxious or experiencing metabolic disorders. Two, the anaerobic threshold (AT) featured an exertion level at which CO2 rates increased and metabolism changed from aerobic to anaerobic. This variable shows individual capacity for exercise and the physiologic responses. Three, respiratory compensation point (RCP) showed the aerobic and anaerobic rates. This is the point when the individual is no longer able to endure lengthy exercise as his or her capacity to remove lactate decreases and the anaerobic system takes over the aerobic system. Four, maximum effort (ME), a marker of cardiopulmonary system limits. Five, recovery (REC) showed heart rate recovery rates, any cardiac stress and the rate of metabolic acidosis after exertion that leads to faster and deeper breathing, as the body tries to eliminate excessive acid by reducing the amount of carbon dioxide (Figure 2). For safety purposes, the experimental environment featured an emergency exit, a team of experts, continuous supervision and monitoring, and an emergency cart equipped with a defibrillator and emergency drugs. Experiment profile for the five stages, environment and study group in each variable (Table 2).

Figure 1 - Test conducted on an underwater treadmill under supervision of a cardiologist.

Figure 2 - Data collected at five moments, both on land and in water.

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0.54 0.49 0.53 0.46 1.02 0.78 1.28 0.99 1.58 1.13 1.71 1.33 2.17 1.60 2.61 1.78 1.15 0.93 1.51 1.11

m SD

0.16 0.17 0.17 0.19 0.37 0.33 0.40 0.23 0.53 0.35 0.50 0.27 0.66 0.33 0.85 0.36 0.26 0.21 0.41 0.24 0.64 0.57 0.59 0.56 1.26 0.97 1.53 1.22 1.69 1.20 1.83 1.44 1.96 1.51 2.35 1.72 0.85 0.68 1.13 0.91

m SD

12.56 13.00 11.49 15.62 13.80 15.45 9.79 17.82 10.74 17.14 8.63 18.48 13.29 20.63 12.77 17.82 12.76 14.12 11.39 22.62

85.90 81.45 88.60 81.30 103.95 90.00 109.10 9.45 122.90 102.40 127.65 108.30 139.35 118.60 149.65 126.50 99.85 89.30 109.15 89.95

0.49 0.82 0.45 0.73 1.05 1.22 1.37 1.60 1.50 1.07 1.62 1.46 1.31 1.35 1.62 1.01 2.72 0.88 1.17 0.83

Land

Pool R

Land

Pool ME

Land

Pool RCP

Land

Pool AT

Land

H DAC H DAC H DAC H DAC H DAC H DAC H DAC H DAC H DAC H DAC Pool

7.15 7.40 7.10 7.30 9.95 10.30 9.25 9.85 12.40 12.10 11.25 12.15 14.85 14.85 15.00 15.20 15.05 15.60 16.10 16.45

Etiology Environment

REP

Stage

Stages: Rest (REP); Aerobic Threshold (AT); Respiratory compensation point (RCP); Maximum effort (ME); Recovery (R); Borg Rating of Perceived Exertion (RPE); Heart Beat (HB). Variables: Oxygen Consumption (VO2); Carbon Dioxide Consumption (VCO2); Oxygen Pulse (VO2/HB); Oxygen Equivalents (VE/VO2); Carbon Dioxide Equivalents (VE/VCO2); Respiratory Exchange Rates (VCO2/VO2). Mean (m); Standard Deviation (SD); Health (H); Coronary artery disease (CAD).

0.08 0.14 0.16 0.12 0.23 0.05 0.11 0.10 0.39 0.10 0.11 0.13 0.15 0.12 0.15 0.12 0.30 0.24 0.42 0.22

SD m

0.85 0.87 0.90 0.84 0.87 0.81 0.84 0.81 1.02 0.94 0.95 0.93 1.09 1.06 1.13 1.04 1.41 1.41 1.37 1.25 6.28 6.39 6.31 7.97 4.03 5.05 3.94 5.85 3.84 4.70 4.10 5.68 4.56 5.02 5.62 5.74 4.93 6.66 5.49 5.82

SD m m SD

2.23 1.92 1.82 2.49 3.16 2.88 3.75 3.21 4.00 2.59 4.06 3.03 3.92 2.86 5.31 4.98 3.17 2.29 3.48 2.67

7.66 7.00 6.66 6.89 12.19 10.80 13.97 13.12 13.65 12.17 14.40 13.62 14.52 12.92 15.90 15.33 8.95 7.13 10.32 10.39

0.14 0.12 0.19 0.14 0.31 0.24 0.35 0.20 0.49 0.33 0.40 0.27 0.71 0.38 0.94 0.31 0.35 0.22 0.53 0.29

VO2/HB VCO2 VO2 HB RPE

Table 2 - Experiment profile for the five stages, by environment and study group in each variable.

30.67 31.82 31.18 30.88 25.00 26.68 24.30 25.92 27.95 30.29 27.77 28.69 34.69 35.27 35.63 34.91 46.41 50.17 45.12 44.05

VE/VO2

36.03 36.09 35.52 35.89 30.81 32.92 29.23 31.77 29.66 32.12 29.30 30.93 31.89 33.30 31.77 33.77 33.10 35.85 33.45 35.20

VE/VCO2

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6.97 9.37 10.86 10.21 3.52 4.05 3.21 5.90 4.17 5.20 4.41 7.22 5.99 6.31 6.20 7.92 10.30 11.41 11.49 11.26

VCO2/VO2

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Statistical Analysis The stage intra-group dependent variables for both on land and in-water tests are RPE, HB, VO2, VCO2, VO2/HB, VE/ VO2, VE/VCO2, and VCO2/VO2. The environment intragroup dependent variable stands for both the on land and in water experiment, and the etiology inter-group independent variables are CAD patients and H individuals. As this was not a treatment in itself, the analogy with a control case experiment was chosen. A variance analysis of repeated measurements (rANOVA) was conducted for factors and for every one of the dependent variables. Time gaps between the stage variables were considered equivalent and were not compromised by the intra-group error term. As non-homogeneous variances were expected throughout the test stages, Greenhouse-Geisser or HuynFeldt corrections (23) were conducted according to the sphericity deviation score, as assessed by the Mauchly test (24). As there is no canonical methodology for identifying significant differences in this type of variance analysis, it was decided to use t-tests for every one of the possible combinations between the three factors under investigation and Holm-Bonferroni corrections for multiple tests. In both cases, the significance level was set at 5%. The sampling population profile is shown by tables with descriptive statistics (means and standard deviations) and is illustrated by graphs of the mean profiles and boxplots for every factor. ANOVA statistical power was calculated at (1 - b)= 0.861, thus totaling a minimum of 20 participants per group.

Ethical Aspects This study was approve by the ethical review board of school of Medicine – University of Sao Paulo, protocol number 0532/08. All subjects agreed to volunteer for the study and signed an informed consent form.

’ RESULTS The analysis showed major effects for CAD group versus Health one for the variables HB, VO2 and VCO2, all of than with (p40.01), no significant effect for the other variables. The major effect for environment, land versus water, showed significance for variables HB, VO2, VCO2 and VO2/HB, all of than with (p40.01) (Table 3). In addition to the major effects described above, it is worth noting that the interactions between factors, particularly the interactions with stage, indicate the subject’s performance throughout the experiment. In this context, the variables RPE Table 3 - Major effects for every variable under investigation. Variables

RPE HB VO2 VCO2 VO2/HB VE/VO2 VE/VCO2 VCO2/VO2

CAD vs Healthy

Land vs Water

2.81 17 12.2 16.5 1.39 0.45 1.35 1.91

0.10 o0.01 o0.01 o0.01 0.25 0.51 0.25 0.17

0 13 25.6 25.4 15.4 1.51 1.76 1.17

1.00 o0.01 o0.01 o0.01 o0.01 0.23 0.19 0.29

RPE-Borg Rating of Perceived Exertion; HB-Heart Beat; VO2-Oxygen Consumption; VCO2-Carbon Dioxide Consumption; VO2/HB-Oxygen Pulse; VE/VO2-Oxygen Equivalents; VE/VCO2-Carbon Dioxide Equivalents; VO2/VO2-Respiratory Exchange Rates; F-Statistics; p-Significance.

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and to make this test as reliable as its counterpart on land. The quality of data depends on several factors, such as subject preparation for capturing ECG signals without any interference using a 5 derivation cable and electrodes covered with occlusive bandages. The major contribution of this study is that it demonstrated that exercise in warm water is well tolerated by patients with CAD and that conducting a CPX in water is feasible and safe. In addition, our results showed that walking or jogging on an underwater treadmill with water at chest level requires less cardiopulmonary exertion than doing so on land. CAD patients showed as high a level of tolerance during the in-water test as the control group subjects, and no adverse events were observed. Issues associated with cardiac preload and post load during immersion were observed as a result of blood displacement, especially from the lower limbs upward to the thoracic cave, thus increasing the volume of blood filling the four heart chambers. Blood volume increases by 27-30% when subjects are immersed to neck level (7). The heart, however, is not a static receptacle and the physiological cardiac response to increased blood volume causes the cardiac muscle to contract more forcefully as the healthy myocardium stretches. According to the Frank and Starling Law (8), contraction efficiency will improve. Gabrielsen et al., 2000 and 1993 (13) showed that both cardiac output and stroke volume increase during immersion in water at 30oC (14). Immersion to the xiphoid process reduces the load on joints and bones by at least 60% (15). The effect of up thrust or buoyancy is an upward vertical force exerted by a fluid and the magnitude of that force is proportional to the weight of the displaced water volume by the object; buoyancy can thus facilitate exercise in water as the force of gravity is reduced. The resistance of the water, however, which is 800 times denser than air, should theoretically require more effort to walk or jog in on an underwater treadmill. Nevertheless, this study shows quite the opposite. As subjects walked or jogged on the underwater treadmill, they did not fight the resistance of the water, and up thrust became a facilitating force as it demanded less exertion of the anti-gravitational muscles to support their body weight. Consequently, the perceived exertion scale Borg RPE showed statistically significant differences between in water and on land tests (po0.01) for both groups in every stage. Christie et al., 1990 (11), demonstrated that the heart rate in healthy subjects has similar responses to exercise on land and in water until VO2 reaches 60% of VO2peak. Our findings demonstrated that the heart rate exhibits significant effects and interactions between etiology and stage, (po0.01), stage and environment (po0.01). At all of the stages, the HB rate was higher on land (p=0.02) than in water (po0.01) for both groups and healthy subjects had a higher HB rate than CAD patients in both environments. This can be explained by the use of medication - CAD group. VO2 seems to be affected by several factors, but the magnitude of oxygen consumption is a much more reliable indicator of cardiorespiratory functional capability (12). In 1989, Gleimand Nicholas (16) demonstrated that oxygen consumption during a walking or jogging exercise at a speed of 53 m/ min was three times greater in water than on land. Therefore, in water, only one-half to one-third of the speed is required to reach the same metabolic intensity rate as on land performance. Because subjects did not run against the resistance of water in this study, this feature might have hindered the collection of similar findings to those in Gleimand Nicholas’

(p40.01), HB (p=0.02), VO2 (p40.01), VCO2 (p40.01), and VO2/HB (p40.01), suggested significant interactions between stage and environment. In other words, the on-land mean profiles were different from the in water profiles throughout the experiment. Moreover, there was a significant interaction between etiology and stage for variables HB (p40.01), VO2 (p40.01), and VCO2 (p40.01). CAD patients and H individuals showed different performances throughout the experiment for these variables. There were no significant interactions regarding the others variables. Furthermore, there was no significant interaction between etiology and environment; these factors can be interpreted independently, which there for e justifies the method chosen for the variance analysis and data interpretation (Table 4). Major effects CAD and Health groups for land versus water, profile for test time measured in minutes and seconds (Figure 3). The Bruce Protocol determines changes in the treadmill incline and speed every 3 minutes, thus resulting in changes in the variables under investigation in the 5 specific set-points of data collection. Hemodynamic changes and cardiovascular overload caused by immersion might lead to a shorter exertion period in the in water test. However, the opposite was observed for both CAD patients and H individuals; the activity time in water was longer than on land (po0.001). ECG changes compatible with myocardial ischemia or arrhythmia were not observed, and the SDBP did not vary significantly.

’ DISCUSSION In this study, we tested the hypothesis that immersion in warm water and the physiological effects associated with exercise during immersion would not be risky for patients with CAD. This study further aimed to investigate and produce reliable and replicable data about cardiorespiratory responses during a CPX conducted in water compared with the test conducted on land. The ‘‘Clinician’s Guide to Cardiopulmonary Exercise Testing in Adults’’ from the American Heart Association (2) clearly states that CPX conducted on land is an extremely safe and versatile test that provides data for evaluating a wide range of responses and levels of tolerance to physical exercise. This set of data is invaluable for the diagnosis and prognosis of patients with cardiovascular diseases. Conducting a CPX in water, however, is not an ordinary practice, as it requires specific equipment, resources and procedures to ensure accurate records and test replicability Table 4 - Interactions between Etiology vs Stage and Environment vs Stage. Variables

RPE HB VO2 VCO2 VO2/HR VE/VO2 VE/VCO2 VCO2/VO2

Etiology x Stage

Environment x Stage

0.32 7.1 6.4 8.17 0.48 0.24 1.59 0.23

0.87 o0.01 o0.01 o0.01 0.75 0.30 0.18 0.92

5.67 3.01 10.1 8.43 6.79 1.72 0.86 1.89

o0.01 0.02 o0.01 o0.01 o0.01 0.15 0.49 0.12

RPE-Borg Rating of Perceived Exertion; HB- Heart Beat; VO2-Oxygen Consumption; VCO2-Carbon Dioxide Consumption; VO2/HB-Oxygen Pulse; VE/VO2-Oxygen Equivalents; VE/VCO2-Carbon Dioxide Equivalents; VO2/VO2-Respiratory Exchange Rates; F-Statistics; p-Significance.

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Figure 3 - Major effects CAD and Health groups for land versus water, profile for test time measured in minutes and seconds.

than in water at all the stages for both CAD and Healthy subjects and both showed lower VO2 in water than on land (po0.01). Craig and Dvorak, 1969 (14), showed that at rest,

study. Nevertheless, our findings show the significant effects and interactions between etiology and stage, (po0.01) and stage and environment (po0.01). VO2 on land was higher

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data collection for the in water test, however, the methodology used for both tests was identical and the subjects had to walk or jog in compliance with the Bruce Protocol (10). The order of the test was not randomized because a CPX in water is not well known and, for safety, we prefer to start with a CPX on land, secure and widely used procedure. This study can be presents a small study population although we performed more 144 tests, but many had to be discarded because of the difficulties in collecting data, primarily in the immersion test. That is why we used the statistical power was calculated at (1 - b)=0.861, thus totaling a minimum of 20 participants per group. This study show that a CPX can be safely conducted in subjects in immersion and that the procedures, resources and equipment used yielded replicable and reliable data. Significant differences observed in water versus on land allow us to conclude that CAD patients are able to do physical exercise in water and that the physiological effects of immersion do not present any risk for such patients, as exercise was well tolerated by all subjects. We can also conclude that CPX conducted in water may be used by cardiologists to safely diagnose and estimate the exertion capability of CAD patients to perform dynamic exercise in a pool. Further investigation is necessary to provide a clearer understanding of the physiological effects of immersion associated with higher risk subjects. A cross-sectional study where the effects of medium and long term are not known, but this was not the objective of the study.

VO2 was similar for healthy subjects on land and in water at 34oC. This finding, however, could not be replicated in the current study, which suggests that the resistance of water or the temperature (34oC) affected oxygen consumption. CO2 production shows a causal relationship with O2 and a linear increase. According to Yazbek et al., 1998 (12), the O2 pulse VO2/ HB indicates the amount of O2 transported during every heart beat. Lower VE performance may be detected in an increasing exertion test by observing the VO2/HB, and it represents an indirect index of O2 transported by the cardiopulmonary system. Significant effects and interactions were observed for stage and environment (po0.01), but not for etiology and stage (p=0.75). According to Hall et al., 1990 (17), a reduced-gravity environment requires less activity of the anti-gravity muscles, which might explain the significant difference observed in the VO2/HB, which was lower in water than on land for both groups (po0.01). However, it does not explain why CAD patients showed lower O2 pulse (po0.01) in water and on land than healthy individuals. The relationships of VE/VO2 and VE/VCO2 with VE at body temperature pressure saturated (BTPS) conditions and VO2 and VCO2 at standard air temperature and air pressure dry (STPD) conditions show how many liters of air per minute are necessary and must be ventilated to consume 100 ml of O2 and to produce CO2. This study has assessed that even during increasing exertion and under the physiological effects of immersion, both CAD patients and H individuals did not show significant changes in VE, which remained appropriate to the relative demand of O2 at each of the stages. In a previous study, Craig and Ware, 1967 (18), and Hall et al., 1990 (19), showed that for healthy individuals, VE decreases during immersion. These findings, however, were not observed in the current study. Our findings corroborate those of Asa C. et al. (20). Ventilatory efficiency during exercise, both on land and in water on a treadmill, was according to the demand of O2. Respiratory quotient or QR reflects the quotient between VCO2 and VO2. During increased exertion, which was the case in this study, metabolism increasingly uses carbohydrates as a source of energy, reaching X1.1 at maximum exertion. In both tests (in water and on land), subjects reached a QR41, so there was no significant effect between stage and environment and etiology and environment. As hemodynamic responses to exercise vary depending on cardiac output and peripheral resistance, increased systolic blood pressure was expected as a result of higher cardiac output in both environments, mainly during immersion. Diastolic blood pressure, in contrast, was expected to remain stable or even to mildly decrease with exercise. However, no significant SDBP response was observed. Studies concerning the SDBP responses of healthy individuals during rest at thermo neutral temperature immersion are conflicting and scarce in the literature Gabrielsen et al., 1993 (13). No significant arrhythmia or ventricular ectopic activity and/or instances of ST-segment depression were observed by the cardiologist in the ECG recordings performance evaluation curve.

’ ACKNOWLEDGMENTS We gratefully acknowledge the support provided by the Institute of Rehabilitation Medicine and LIM 01 - Clinics Hospital - School of Medicine University of Sao Paulo and Dante Pazzanese Institute of Cardiology, Sao Paulo, Brazil. This work was partially supported by grants provided by LIM01-HCFMUSP, CNPq, Institute of Rehabilitation and Medicine Clinics Hospital - School of Medicine - University of São Paulo.

’ AUTHOR CONTRIBUTIONS Garcia MK was responsible for the study conception and design, analysis and interpretation of data and manuscript writing. Rizzo L was responsible for the coordination of the research, conduction of the experiments and manuscript writing. Yazbek-Júnior P was responsible for the conduction of the experiments with cardiopulmonary stress exertion analysis and interpretation of data. Yutiyama D, Silva F.J and Matheus D were responsible for the conduction of the experiments. Mastrocolla LE was responsible for the analysis and interpretation of the ﬁndings. Massad E was responsible for the study conception, design, analysis and interpretation of the ﬁndings.

’ REFERENCES 1. Schmid JP, Noveanu M, Morger C, Gaillet R, Capoferri M, Anderegg M, et al. Influence of water immersion, water gymnastics and swimming on cardiac output in patients with heart failure. Heart. 2007;93(6):722-7, http://dx.doi.org/10.1136/hrt.2006.094870. 2. Balady GJ, Arena R, Sietsema K, Myers J, Coke L, Fletcher GF, et al. Clinician’s Guide to cardiopulmonary exercise testing in adults: a scientific statement from the American Heart Association. Circulation. 2010; 122(2):191-225, http://dx.doi.org/10.1161/CIR.0b013e3181e52e69. 3. Norsk P, Bonde-Petersen F, Walberg J. Central venous pressure and plasma arginine vasopressin in man during water immersion combined with changes in blood volume. Eur J Appl Physiol Occup Physiol. 1986; 54(6):608-16, http://dx.doi.org/10.1007/BF00943349. 4. Arborelius M Jr, Ballidin UI, Lilja B, Lundgren CE. Hemodynamic changes in man during immersion with the head above water. Aerosp Med. 1972;43(6):592-8.

Study limitations The use of an underwater treadmill without any computer interface prevented this study from automatically controlling the treadmill speed and incline. This might have affected

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15. Harrison RA, Bulstrode S. Loading of the lower limb when walking partially immersed.Physiotherapy. 1992;78(3):164-6, http://dx.doi.org/ 10.1016/S0031-9406(10)61377-6. 16. Gleim GW, Nicholas JA. Metabolic costs and heart rate responses to treadmill walking in water at different depths and temperatures. Am J Sports Med. 1998;17(2):248-52, http://dx.doi.org/10.1177/036354658901700216. ́ 17. Hall J, Macdonald IA, MaddisonPJ, OHare JP. Cardiorespiratory responses to underwater treadmill walking in healthy females. Eur J Appl Physiol Occup Physiol. 1998; 77(3):278-84, http://dx.doi.org/10.1007/ s004210050333. 18. Craig AB Jr, Ware DE. Effect of immersion in water on vital capacity and residual volume of́ the lungs. J Appl Physiol. 1967;23(4):423-5. 19. Hall J, Bisson D, OHare P. The physiology of immersion. Physiotherapy. 1990;76(9):517-21, http://dx.doi.org/10.1016/S0031-9406(10)63019-2. 20. Cider A, Sunnerhagen KS, Schaufelberger M, Andersson B. Cardiorespiratory effects of warm water immersion in elderly patients with chronic heart failure. Clin Physiol Funct Imaging. 2005;25(6): 13-7, http:// dx.doi.org/10.1111/j.1475-097X.2005.00633.x. 21. Brandão MR, Pereira MH, Oliveira R, Matsudo VK. Percepc¸ão de esforc¸o: uma revisão da área. Revista Brasileira de Ciências do Movimento. 1989; 3(1):34-40. 22. The Criteria Committee of the New York Heart Association. Nomenclature and Criteria for Diagnosis of Diseases of the Heart and Great Vessels. (9th ed.). Boston: Little, Brown & Co. 1994; 253–256. 23. Greenhouse SW, Geisser S. On methods in the analysis of profile data. Psychometrika. 1959;24(2):95-112, http://dx.doi.org/10.1007/BF02289823. 24. Mauchly, JW. Significance Test for Sphericity of a Normal n-Variate Distribution. The Annals of Mathematical Statistics. 1940;11(2): 204-9, http://dx.doi.org/10.1214/aoms/1177731915.

5. Risch WD, Koubenec HJ, Beckmann U, Lange S, Gauer OH. The effect of graded immersion on heart volume, central venous pressure, pulmonary blood distribution, and heart rate in man. Pflugers Arch. 1978;374(2): 115-8, http://dx.doi.org/10.1007/BF00581289. 6. Lange L, Lange S, Echt M, Gauer OH. Heart volume in relation to body posture and immersion in a thermo-neutral bath. A roentgenometric study. Pflügers Arch. 1974;352(3):219-26, http://dx.doi.org/10.1007/BF00590487. 7. Weston CF, O’Hare JP, Evans JM, Corrall RJ. Hemodynamic changes in man during immersion in water at different temperatures. Clin Sci (London). 1987;73(6):613-6, http://dx.doi.org/10.1042/cs0730613. 8. Becker BE, Cole AJ. Aquatic rehabilitation. In: DeLisa JA (eds). Physical Medicine and rehabilitation: principles and practice. 4th ed. Philadelphia: Lippincott. 2005;479-92. 9. Epstein M. Renal effects of head-out water immersion in humans: a 15-year update. Physiol Rev. 1992;72(3):563-621. 10. Bruce RA, Pearson R, Lovejoy Jr FW, Yu PN, Brothers GB. Variability of respiratory and circulatory performance during standardized exercise. J Clin Invest. 1949;28(6 Pt 2):1431-8, http://dx.doi.org/10.1172/JCI102208. 11. Christie JL, Sheldahl LM, Tristani FE, Wann LS, Sagar KB, Levandoski SG, et al. Cardiovascular regulation during head-out water immersion exercise. J Appl Physiol. 1990;69(2):657-64. 12. Yazbek-Jr P, Carvalho RT, Sabbag LM, Battistella LR. Ergoespirometria. Teste de Esforc¸o Cardiopulmonar, Metodologia e Interpretac¸ão. Arq Bras Cardiol. 1998;71(5):719-24, http://dx.doi.org/10.1590/S0066-782X199800 1100014. 13. Gabrielsen A, Johansen LB, Norsk P. Central cardiovascular pressures during graded water immersion in humans. J Appl Physiol. 1993;75(2):581-5. 14. Craig A, Dvorak M. Comparison of exercise in air and in water of different temperatures. Med Sci Sports. 1969;1:124-30.

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CLINICAL SCIENCE

Impact of the delay to start treatment in patients with lung cancer treated in a densely populated area of Brazil Fernando Conrado Abrao,I,II,* Igor Renato Louro Bruno de Abreu,I,II Roberto Odebrecht Rocha,I Felipe Dourado Munhoz,I Joa˜o Henrique Godoy Rodrigues,I Riad Naim YounesII I Departamento de Cirurgia Toracica, Faculdade de Medicina Santa Marcelina, Sao Paulo, SP, BR. II Departamento de Cirurgia Toracica, Centro de Oncologia do Hospital Alemao Oswaldo Cruz, Sao Paulo, SP, BR.

OBJECTIVES: The aim of this study is to evaluate the access of patients with lung cancer in a densely populated area of Sa˜o Paulo to the Brazilian Public Health System, focusing on the time spent from symptom onset or initial diagnosis until the beginning of treatment. METHODS: We retrospectively reviewed 509 patients with malignant lung neoplasms who were admitted to a single reference oncology center of the public health system between July 2008 and December 2014. Patients were considered eligible for this study if they were older than 18 years and had not undergone any previous oncology treatment when they were admitted to the institution. The following data were collected from all patients: age, gender, smoking status, tumor staging, time from the when the first symptoms were experienced by the patient to when the patient was diagnosed with cancer, time from the first appointment to cancer diagnosis, and time from when the patient was diagnosed with cancer to the initiation of treatment. RESULTS: The median time from symptom onset to diagnosis was three months. From the first appointment to diagnosis, the median time interval was one month; however, 79% of patients were diagnosed in up to two months. The median time from diagnosis to the start of treatment was one month, but most patients (82.5%) started treatment in up to two months. CONCLUSION: In our highly populated region with preferential access to the public health system, patients are required to wait a relatively long time to effectively begin treatment for lung cancer. This type of study is important to alert medical societies and government health agencies. KEYWORDS: Lung Neoplasms; Public Health; Mortality. Abrao FC, Abreu IR, Rocha RO, Munhoz FD, Rodrigues JH, Younes RN. Impact of the delay to start treatment in patients with lung cancer treated in a densely populated area of Brazil. Clinics. 2017;72(11):675-680 Received for publication on May 23, 2017; First review completed on June 7, 2017; Accepted for publication on August 8, 2017 *Corresponding author. E-mail: fernandocabrao@uol.com.br

’ INTRODUCTION

to an estimated risk of 16.79 new cases per 100,000 men and 10.75 per 100,000 women. Excluding non-melanoma skin tumors, lung cancer in men is the second most common malignancy in the southern (35.17 / 100,000) and midwestern (14.53 / 100,000) regions of Brazil. In the southeastern (19.02 / 100,000), northeastern (9.75 / 100,000) and northern (8.07 / 100,000) regions of Brazil, lung cancer is the third most common cancer among men. For women, lung cancer is the third most common malignancy in the southern (20.61 / 100,000) and southeastern (10.56 / 100,000) regions. In the midwestern (9.37 / 100,000) and northeastern (7.24 / 100,000) regions of Brazil, lung cancer is the fourth most common malignancy in women. In the northern region (5.07 / 100,000), lung cancer is the fifth most common among women (5). Unlike the data from developed countries, the lung cancer incidence and mortality in Brazil follow an upward curve, with progressively higher diagnostic and mortality rates. (6). In 1992, Pereira et al. described the profile and access of lung cancer patients in the Brazilian Public Health System (SUS). The author showed data related to the initial staging and warned about the delay in the diagnostic process (7). However, this

Lung cancer is the malignant neoplasm with the highest incidence worldwide (1,2). In developed countries, the incidence has decreased in men and remained stable among women, reflecting a reduction in the rate of smoking in males (3). Lung cancer is also the leading cause of cancer-related deaths worldwide, with an estimated 1.1 million deaths in men and 427,400 in women in 2012 (4). These numbers are greater than the sum of deaths due to breast, prostate and colon cancers and account for approximately 18% of deaths from all cancers (1). In 2016 in Brazil, 17,490 new cases of lung cancer were estimated in men and 10,540 in women. These values correspond

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b) Time (months) from the date of the first appointment (first app) with a specialist (oncologist or thoracic surgeon) to the date when the patient was diagnosed with cancer (DX); we excluded seven patients from whom we did not recover this information when we conducted this specific analysis. c) Time (months) from the DX date to the starting date of treatment (TTO).

study did not assess the necessary time to start treatment. After this study, the literature over the past few decades did not include another study that assessed the evolution of health care access of lung cancer patients in the SUS. The aim of this study is to evaluate the recent access (in the last six years) of lung cancer patients to the SUS, including the time required to start treatment.

’ METHODS

Statistical Analyses

This study is part of a research project approved by the Ethics Review Committee at our institution, registered under protocol 49258615.4. We retrospectively reviewed 509 patients with malignant lung neoplasms who were admitted to a single reference oncology center of the public health system between July 2008 and December 2014, which were our inclusion criteria. The median follow-up time was seven months (range 1 – 77 months). Our institution is one of the reference oncology centers in the state of São Paulo, which is the most densely populated area of Brazil. Our center is recognized by the executive board of the public health system as an institution able to treat any type of cancer, including pediatric cancer. Patients diagnosed with lung cancer during the study period were identified by the Cancer Registry database of our institution. Additional clinical information on individual patients was collected retrospectively from medical records kept at our institution. All patients admitted were originally from public health care units (general practitioners or pulmonologists) and required a recommendation to the oncology center from the executive board of the public health system. The board of the public health system refers these patients to one of the reference oncology centers. Patients were considered eligible for this study if they were older than 18 years and had not undergone any previous oncology treatment when they were admitted. All 509 patients were eligible for this study. We did not find any patients who had undergone previous oncology treatment or patients younger than 18 years old. Histopathological exams were registered to confirm lung cancer diagnoses. All patients admitted to our institution without histopathological exams were submitted to tumor biopsy guided by computed tomography (CT), ultrasound or bronchoscopy. Patients with suspected stage I lung cancer were subjected to a CT scan that included the thorax, abdomen and pelvis. Patients with suspected stage II or more advanced disease were subjected to positron emission tomography (PET) and brain nuclear magnetic resonance imaging. Patients who were candidates for surgical treatment were subjected to a frozen biopsy of the lung tumor and the mediastinal lymph nodes in the operating room. The following data were collected from all patients: age range, gender, smoking status and tumor staging according the American Joint Committee on Cancer and the International Union for Cancer Control update of the tumor-node-metastasis (TNM) cancer staging system (8,9). In this study, we also evaluated the influence of time until diagnosis and time to initiate the treatment on the survival of patients with lung cancer. The data were collected and arranged in the following time intervals:

Descriptive analysis of the data (i.e., distribution and the presence of outliers) was carried out using measures of central tendency (median and interquartile range), measures of dependence (correlation and covariance) and measures of dispersion (amplitude and/or standard deviation) for continuous variables and frequency for categorical variables. Categorical and continuous variables were compared by Fisher’s exact test or by chi-squared and Mann-Whitney U tests, respectively. In addition, measures of skewness and a coefficient of kurtosis were evaluated. Death related to lung cancer was the principal outcome evaluated in this study. Kaplan-Meier survival estimates were used to determine the five-year lung cancer-specific survival for all patients, and the log-rank (Mantel-Cox method) and Breslow (generalized Wilcoxon) analyses were used to compare the differences between factors. The cutoff values were chosen based on medical literature regarding delayed treatment in lung cancer (10-12). Survival was calculated from the date of patient admission to our institution to the date of the last follow-up or until death from any cause. The data are presented as the medians and 95% confidence intervals (CIs; lower – upper bounds). Univariate and multivariate Cox proportional hazard analyses were performed to investigate the prognostic significance of the demographic and predictive risk factors on the survival outcome of patients with lung cancer. Statistical analyses were performed using SPSS version 17.0 for Windows. A two-tailed value of p o 0.05 indicated statistical significance.

’ RESULTS The demographic characteristics of the 509 lung cancer patients are summarized in Table 1. The mean age of these patients was 62.5 years (standard deviation: 11.2 years), and there was a slight predominance of male (57.8%) over female (42.2%) patients. More than 75% of these patients were smokers. Lung adenocarcinomas (37.3%) and squamous cell carcinoma (28.7%) were the most common types of tumors observed in the patients, and carcinoid tumors (2.1%) were the least frequent type. Regarding clinical stages, most patients (68.3%) came to our hospital when the disease was already at an advanced stage (stages III and IV). Most of these patients began receiving treatment at up to 1.5 months after DX (Table 2). Approximately one-fifth of the patients were eligible for surgery (20.2%), and lobectomy (67.0%) was the most frequently performed surgical procedure. For all patients, including those who received no cancer-specific treatment, the median overall survival was seven months (95% CI: 5.7 to 8.2 months), with 34.5% of these patients surviving for one year and 8.1% surviving for five years. Patients spent a relevant amount of time waiting during each interval period. For instance, the median time from PH to DX was 3 months. From the first app to DX, the median

a) Time (months) from the date when the first symptoms were experienced by the patient (patient history, PH) to the date when the patient was diagnosed with cancer (DX); we excluded 37 patients who were asymptomatic when we conducted this specific analysis.

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Table 1 - Demographic characteristics of the 509 lung cancer

Table 2 - Stage of lung cancer with respect to time (months) from the date when the patient was diagnosed with cancer (DX) to the starting date of treatment (TTO), showing the number of patients at each stage according to the different delays*.

patients. Parameters Patient age (mean±SD), years Number of patients Gender Male Female Histology type of primary tumor Adenocarcinoma Large cell carcinoma Small cell lung cancer Squamous cell carcinoma Undefined Stages of cancer Ia/Ib IIa/IIb IIIa/IIIb IV Extensive a Limited b Undefined Surgical procedure Yes No Surgical procedure type Bi-lobectomy Lobectomy Pneumectomy Segmentectomy Thoracotomy Disease recurrence Yes No Smoker Yes No Cigarette packs per year (mean ± SD)

Total Number

(62.5±11.2) 509

100%

294 215

57.8% 42.2%

187 11 24 74 146 67

37.3% 2.1% 4.7% 14.5% 28.7% 13.2%

37 28 74 274 58 15 23

7.3% 5.5% 14.5% 53.8% 11.4% 2.9% 4.5%

103 406

20.2% 79.8%

1 69 15 9 9

1.0% 67.0% 14.5% 8.7% 8.7%

127 382

24.9% 75.1%

400 109

78.6% 21.4%

Stage of lung cancer

Ia/Ib IIa/IIb IIIa/IIIb IV Extensive Limited Undefined

Number of patients (%)

Delay between DX and TTO p1.5 months

Delay between DX and TTO 41.5 months

Total 509 (100) 37 28 74 274 58 15 23

Total 334 (65) 26 (70) 14 (50) 46 (62) 177 (65) 40 (69) 08 (53) 22 (96)

Total 175 (35) 11 (30) 14 (50) 28 (38) 94 (35) 18 (31) 07 (47) 01 (04)

* p value = 0.039 (Fisher’s exact test, p1.5 vs. 41.5 months).

These results were confirmed in the multivariate analysis (Table 3).

’ DISCUSSION In Brazil, to the best of our knowledge, one study has assessed the time from diagnosis to treatment of lung cancer (7). In 1992, Pereira et al. studied patients at all stages and not only patients who were candidates for surgical resection (7). Our study data, obtained 23 years later, suggest that the time required for lung cancer diagnosis has decreased. The data also suggest that diagnosis is faster when the patient can make an appointment with a specialist (i.e., a pulmonologist, thoracic surgeon or oncologist). Additionally, as far as we know, this is the first study that evaluated the time to begin cancer treatment or palliative care across all cancer stages in the public health system. Other positive results on the survival from this study are the cancer stage, histological type of lung cancer and surgical treatment. However, these results are well stablished in the medical literature. Pereira et al. reported that only 44.3% of patients were diagnosed with lung cancer within 90 days. On the other hand, 45.5% of patients were diagnosed after 120 days. However, the same study showed that access to the public health system was fast, with 88% of patients arriving at the first consultation within 30 days after symptom onset, but only 10% were initially seen by a specialist in the oncopulmonology area. Our study showed that the median time from PH to DX was 90 days, and 79% of cases had a lung cancer diagnosis confirmed within 60 days. Another important finding is that after the first consultation with a specialist, the median time to diagnosis was 30 days. These data suggest that in the last 23 years, there was a decrease in the time to diagnosis of lung cancer and that medical specialists, not generalists, played an important role in accelerating the time to diagnosis. In the United States, Nadpara et al. reported a median time between first symptomatic presentation and diagnosis of approximately 180 days (13). A median diagnostic interval of 113 days (with an upper quartile value of 249 days) was reported in a recent English primary care records study of lung cancer patients diagnosed between 2007 and 2010 (14). However, regarding the time required to start treatment after diagnosis, 50% of patients required up to 30 days, 32.5% required between 30 and 60 days, and 17.5% required more

(50.5±32.9)

Extensive disease: Beyond the ipsilateral hemithorax, which may include malignant pleural or pericardial effusion or hematogenous metastases (TNM: T any, N any, M Ia/b; T3-T4 due to multiple lung nodules that do not fit in a tolerable radiation field; 9). b Limited disease: Confined to the ipsilateral hemithorax, which can be safely encompassed within a tolerable radiation field (TNM: T any, N any, M 0; except T3-T4 due to multiple lung nodules that do not fit in a tolerable radiation field; 9).

time interval was 1 month; however, 79% of patients were diagnosed in up to 2 months. Finally, the median time from DX to TTO was 1 month (range: 0 – 17 months), but most patients (82.5%) started treatment in up to 2 months. The effects of the time intervals from the development of the first symptoms to TTO on lung cancer-associated mortality are shown in the set of Kaplan-Meier survival curves using the log-rank and/or Breslow statistical tests. A KaplanMeier curve demonstrated a significant negative impact on the cumulative five-year survival curve for patients who started treatment up to 1.5 months after DX. The time from PH to DX and the time from the first app with the general practitioner to DX were not statistically significant (Figures 1, 2 and 3).

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Figure 1 - Kaplan Meier curve for mortality due to lung cancer in patients according to time from the patient history (PH) to diagnosis (DX). The blue line represents the patients diagnosed up to 3 months, and the green line represents patients diagnosed above 3 months.

Figure 2 - Kaplan Meier curve for mortality due to lung cancer in patients according to time from the appointment (first app) to diagnosis (DX). The blue line represents the patients diagnosed up to 2 months and green line above 2 months.

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Figure 3 - Kaplan Meier curve for mortality due to lung cancer in patients according to time from diagnosis (DX) to the starting date of treatment (TTO). The blue line represents the patients that started treatment above 1.5 months and green line up to 1.5 months.

Table 3 - Risk factors (prevalence odds ratio, 95% CI) associated with patient death by univariate and multivariate analyses (Cox regression). Risk factors, N (%)

Multivariate analysis p value

Univariate analysis

Multivariate analysis

Age 4 62 years Gender Male

1.1 (0.9 – 1.3) 1.1 (0.9 – 1.4)

not included not included

Histology type of primary tumor Adenocarcinoma Large cell carcinoma Squamous cell carcinoma Small cell lung cancer

Comparator 1.3 (0.8 – 2.2) 1.3 (1.0 – 1.6) 2.3 (1.7 – 3.2)*

2.0 (1.5 – 2.8)

o0.001

Stages of cancer Ia/Ib IIa/IIb IIIa/IIIb IV Extensive Limited

comparator 1.6 (0.8 – 3.2) 2.7 (1.6 – 4.7)* 3.7 (2.2 – 6.2)* 3.9 (2.2 – 6.9)* 2.7 (1.3 – 5.6)*

2.5 (1.4 – 4.6) 3.0 (1.7 – 5.4) -

0.002 o0.001 ns ns ns

No surgical treatment for lung cancer Surgical treatment for lung cancer Time from PH to DX 4 3 months Time from first app to DX 4 2 months Time from DX to TTO o 1.5 months

comparator 0.4 (0.3 – 0.6)* 1.0 (0.8 – 1.2) 1.2 (1.0 – 1.5) 1.8 (1.4 – 2.2)*

0.6 (0.4 – 0.9) not included not included 2.1 (1.6 – 2.6)

0.005

o0.001

* p value o0.05 (univariate analysis by Cox regression).

the number of patients who waited more than 30 days to start treatment is substantial. Regarding the decrease in survival observed in patients who started treatment up to 1.5 months after diagnosis, Myrdal et al. found the same result (11). In both studies, the number of patients in clinical stages III and IV was higher in the group treated early (Table 2). Therefore, in agreement

than 60 days. On the other hand, in South Korea, Shin et al. described only the surgical delay for patients with lung cancer, and the median time from cancer diagnosis to surgery was 20 days (15). In Finland, for all patients with lung cancer, the median time from cancer diagnosis to treatment was 15 days (10). Our study did not assess the possible causes that could explain our results. However, it is noteworthy that

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with Myrdal et al., we believe that more severe patients, i.e., those at more advanced stages, end up being prioritized to start treatment sooner than those at lower stages. The authors of both studies believe that this phenomenon reflects the health system’s incapacity to absorb all patients. Perhaps this deficiency of the public health system could be minimized with the implementation of early detection programs for lung cancer. Our study has several limitations. This is a retrospective study of data from a single reference oncology center. Mortality data should be interpreted with caution, as the study included patients who received exclusively palliative treatment as well as patients with different types of biological tumors (e.g., small cell carcinoma). We conclude that there was a reduction in the time to lung cancer diagnosis over the past two decades. Access to a specialist plays a crucial role in diagnosis in the public health system, and in our densely populated area, patients have to wait a relatively long time to begin treatment. This type of study is important to alert medical societies and government health agencies.

2. No authors listed. Atlas of cancer mortality in the European Union and the European Economic Area 1993-1997. IARC Sci Publ. 2008;(159):1-259. 3. Siegel R, Naishadam D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin. 2012;62(1):10-29, http://dx.doi.org/10.3322/caac.20138. 4. American Cancer Society. Global Cancer Facts & Figures 3rd edition. Atlanta: American Cancer Society;2015. 5. Instituto Nacional de Câncer / Ministério da Saúde. Estimativa 2016: incidência de câncer no Brasil. Rio de Janeiro: INCA. http://www.inca. gov.br. (acesso em 07/04/2016). 6. Araujo LH, Baldotto CS, Zukin M, Vieira FM, Victorino AP, Rocha VR, et al. Survival and prognostic factors in patients with non-small cell lung cancer treated in private health care. Rev Bras Epidemiol. 2014;17(4):1001-14, http://dx.doi.org/10.1590/1809-4503201400040017. 7. Silva PP, Pereira JR, Ikari FK, Minamoto H. Lung cancer and the delay in the diagnosis: analysis of 300 cases. Rev Assoc Med Brasil (1992):38(3): 145-9. 8. Edge SB, Compton CC. The American Joint Committee on Cancer: the 7th edition of the AJCC cancer staging manual and the future of TNM. Ann Surg Oncol. 2010;17(6):1471-4, http://dx.doi.org/10.1245/s10434010-0985-4. 9. NCCN Clinical Practice Guidelines in Oncology: Small Cell Lung Cancer V 2.2013. Available at http://www.nccn.org/professionals/physician_gls/ pdf/sclc.pdf. Accessed: May 12, 2016. 10. Salomaa E, Sallinen S, Hiekkanen H, Liippo K. Delays in the diagnosis and treatment of lung cancer. Chest. 2005;128(4):2282-8, http://dx.doi. org/10.1378/chest.128.4.2282. 11. Myrdal G, Lambe M, Hillerdal G, Lamberg K, Agustsson T, Stahle E. Effect of delays on prognosis in patients with non-small cell lung cancer. Thorax. 2004;59(1):45-9. 12. Hueto Pérez De Heredia J, Cebollero Rivas P, Cascante Rodrigo JA, Andrade Vela I, Pascal Martínez I, Boldú Mitjans J, et al. Evaluation of the use of a rapid diagnostic consultation of lung cancer. Delay time of diagnosis and therapy. Arch Bronconeumol. 2012;48(8):267-73. 13. Nadpara P, Madhavan SS, Tworek C. Guideline-concordant timely lung cancer care and prognosis among elderly patients in the United States: A population-based study. Cancer Epidemiol. 2015;39(6):1136-44, http:// dx.doi.org/10.1016/j.canep.2015.06.005. 14. Din NU, Ukoumunne OC, Rubin G, Hamilton W, Carter B, Stapley S, et al. Age and Gender Variations in Cancer Diagnostic Intervals in 15 Cancers: Analysis of Data from the UK Clinical Practice Research Datalink. PLoS One. 2015;10(5):e0127717, http://dx.doi.org/10.1371/ journal.pone.0127717. 15. Shin DW, Cho J, Kim SY, Guallar E, Hwang SS, Cho B, et al. Delay to curative surgery greater than 12 weeks is associated with increased mortality in patients with colorectal and breast cancer but not lung or thyroid cancer. Ann Surg Oncol. 2013;20(8):2468-76, http://dx.doi.org/ 10.1245/s10434-013-2957-y.

’ AUTHOR CONTRIBUTIONS Abrao FC designed the study, analyzed the data, wrote the manuscript, reviewed the analysis of the data and approved the final version of the manuscript. Abreu IR reviewed the analysis of the data and approved the final version of the manuscript. Rocha RO analyzed the data and reviewed the statistical analysis. Munhoz FD and Rodrigues JG reviewed the analysis of the data and helped to conduct the study. Younes RN helped to design the study, analyzed the data and helped to write the manuscript. All the authors read and approved the final version of the manuscript.

’ REFERENCES 1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global Cancer Statistics. CA Cancer J Clin. 2011;61(2):69-90, http://dx.doi.org/10.3322/ caac.20107.

680

CLINICAL SCIENCE

Dental status, oral prosthesis and chewing ability in an adult and elderly population in southern Brazil Alexandre Baumgarten,I Jeanne Gabriele Schmidt,I Rafaela Soares Rech,I Juliana Balbinot Hilgert,I,II Ba´rbara Niegia Garcia de GoulartI,* I Graduate Program in Epidemiology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil. II Graduate Program in Dentistry, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.

OBJECTIVE: The objective of this study was to explore the factors associated with inadequate chewing in an adult and elderly population of a city in the southern region of Brazil. METHODS: This was a cross-sectional study based on a population home-based inquiry (DCH-POP) in southern Brazil. Individuals were interviewed by trained interviewers to create a standardized procedure. In a pilot study, the Questionnaire of Human Communication Disorders (DCH-POP) was created and validated to identify selfreported speech and language, swallowing and hearing disorders. The outcome was dichotomized into either having adequate chewing or not, as assessed by a series of questions about chewing ability. Analyses of absolute and relative frequencies were measured according to the studied variables. A Poisson regression was applied at a significance level of 5%. RESULTS: A total of 1,246 people were interviewed. Inadequate chewing was found in 52 (5.6%) individuals, with a higher prevalence in the elderly (11.8%) than in adults (5.2%). In the final model, the following factors were associated with inadequate chewing: being 61 years of age or older (prevalence ratio or PR=9.03; 95% CI: 1.20-67.91), loss of teeth and use of unadjusted prosthesis (PR=3.50; 95% CI: 1.54-7.95), preference for foods of soft consistency (PR=9.34; 95% CI:4.66-8.70) and difficulty in nasal breathing (PR=2.82; 95% CI: 1.31-6.06). CONCLUSION: Age, oral health status through dental prosthesis, preference for foods of soft consistency and difficulty breathing through the nose were factors associated with chewing inability in adults and the elderly. KEYWORDS: Adult; Aged; Chewing Ability; Oral Health; Dental Prosthesis. Baumgarten A, Schmidt JG, Rech RS, Hilgert JB, de Goulart BN. Dental status, oral prosthesis and chewing ability in an adult and elderly population in southern Brazil. Clinics. 2017;72(11):681-685 Received for publication on April 19, 2017; First review completed on July 23, 2017; Accepted for publication on August 9, 2017 *Corresponding author. E-mail: bngoulart@gmail.com

’ INTRODUCTION

are an artificial substitute for the teeth and may perform a similar function, the use of dental prostheses and/or unadjusted prostheses does not perform a satisfactorily masticatory function, which leads to changing eating habits (3,8). In some cases, chewing occurs in conjunction with oral breathing, whether or not it is associated with nasal obstruction. In these cases, the activity of the masticatory muscles and the time spent to perform the function are reduced. Breathing through the mouth may reduce the degree and duration of the vertical occlusal force on the posterior teeth and may induce vertical problems in malocclusion (9,10). Thus, prolonged chewing is required to form a bolus and initiate swallowing. Oral breathing restricts chewing in daily life, and it is not uncommon that chewing competes with breathing and may be associated with chewing inefficiency, as well as dentofacial alterations, at any stage of life (11,12). Chewing, therefore, plays a critical role in the daily lives of all individuals. Previous studies examining this phenomenon usually use clinical or institutionalized populations (8,10,11,13). It is critical to understand the distribution of chewing ability as well as the factors associated with this condition through

Chewing involves neuromuscular and digestive activities. It is considered the most important function of the stomatognathic system (1)and indicates the ability to crush, grind and mix food with saliva, as well as the ability to form the bolus (2). Thus, the act of chewing creates a relation of interdependence with nutritional conditions, since impaired chewing can decrease nutritional quality (3,4). Tooth losses influence chewing function and efficiency (5,6). It has been established that tooth loss is associated with the election of food consistency, difficulty in food deterioration, and poorer chewing ability (7). Although dental prostheses

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information on the following: I) tooth loss; II) use of dental prosthesis (has all teeth, lost teeth and does not use prosthesis, lost teeth and uses fitted prosthesis, or lost teeth and uses unadjusted prosthesis); III) keep their mouth open most of the time; IV) previous speech therapy treatment; V) preference for a specific type of food consistency; and VI) difficulty with nasal breathing.

a population-based household survey to understand the dysfunctions in the adult and elderly population, such as difficulties in eating consistent foods and difficulties in forming the food bolus for swallowing (14,15). It is also critical to have subsidies to propose actions directed towards health promotion and the greatest effectiveness in the scope of prevention, diagnosis and rehabilitation. Thus, the objective of this study was to explore the self-reported factors associated with inadequate chewing in an adult and elderly population of a city in southern Brazil.

Data analysis

Data were analysed using SPSS v.21 software (Chicago: SPSS Inc). Absolute and relative frequency analyses were calculated, in addition to chi-square tests, independent samples t-tests and Fisher’s exact tests. A po0.05 significance level was used to evaluate differences in the studied variables. A Poisson regression with robust variance was performed to obtain the prevalence ratios (PR) with their respective 95% confidence intervals (95% CI). Three models were created to test the associations between the outcome and the selfreported associated factors.

’ METHODS Type of study and sample qualification A cross-sectional population-based study was conducted in the city of Porto Alegre, Brazil, between 2010 and 2014. This study is part of a household survey of the self-reported Human Communication Disorders Population Study (HCDPOP) (16). Probabilistic sampling was performed, stratified by multiple stages and determined from an analysis of age distribution and education. For the sample size of 1,500 individuals, a significance level of 95% was used to establish the confidence intervals (z=1.96), with a sampling error of 10% and a proportion of 20% (p=0.20) to be estimated in population subgroups. The absolute number of people with human communication disorders was estimated by expanding the sample data to the total Brazilian population in the same age group and geographic area (16). Differences between categories were assessed by the overlap of their confidence intervals. The criterion of eligibility was to reside at an address in the selected neighbourhood. After selecting the residences, buildings with multiple units had their individuals listed from the lowest floor to the highest floor. All residents in the selected houses were considered eligible for the study, with exclusionary factors of being institutionalized during the collection of data, the occurrence of four or five home visits at alternate times with no answer, as well as unsuccessful telephone contact. One respondent per household was chosen according to his or her willingness to participate in the survey. For the data collection, interviewers were selected and given uniform training and face-to-face refreshment courses every three weeks to help them recall the methodology of all data collection steps, with the objective of monitoring data collection and improving respondents’ likelihood of participating in the study.

Ethical research criteria This study was approved by the Research Ethics Committee of the Federal University of São Paulo under number 0150/2010. The researchers followed the guidelines set forth in Resolution 496 by the National Health Council.

’ RESULTS Out of the 1,500 subjects predicted in the HCD-POP study, 1,246 individuals were interviewed (losses and refusals: 16.9%), 321 of whom were excluded from this analysis because they were under 18 years old. The other 925 participants corresponded to the population of this study. Women constituted the majority of the sample (58.1%). The mean age was 48.9 (SD ±19.6) years, while the number of years of education was 12.9 (SD ±3.4) years. Inadequate chewing was found in 52 (5.6%) individuals in the sample and was more prevalent in the elderly (11.8%) than in adults (5.2%). The proportion of AC in the sample and its associations with socioeconomic, orofacial and anatomical characteristics are presented on Table 1. No statistically significant associations (p40.05) were found between AC and sex or education. IC was more frequently found in older individuals (mean age of 67.2 (SD ±16.5) years) than those who had AC, which was found in individuals with a mean age of 47.8 (SD ±19.3) years (po0.001). In contrast, IC was self-reported more often in individuals who had lost their teeth (65.4%) and who wore dental prostheses (61.5%) (po0.001). As the age of the individuals in the sample increases, an increase in inadequate chewing and number of disorder alterations is also observed (Figure 1). Table 2 presents a Poisson regression with crude and adjusted prevalence ratios and confidence intervals. After adjusting the final model, some independent variables lost their statistical significance, remaining associated with the outcome only: being 61 years of age or older (PR=9.03; 95% CI: 1.20-67.91), loss of teeth and unadjusted prosthesis (PR=3.50; 95% CI: 1.54-7.95), preference for foods of soft consistency (PR=9.34; 95% CI:4.66-18.70) and difficulty with nasal breathing (PR=2.82; 95% CI: 1.31-6.06). The adjusted model was verified by the w2 Pearson test (p=0.864) and omnibus test (po0.001). Subgroup analysis was performed, but no effect modification was identified.

Outcomes and variables The outcomes included issues related to chewing and its possible difficulties, dichotomized into either having adequate chewing (AC) or not (inadequate chewing or IC). The chewing was considered adequate upon a negative response to the following items: chewing difficulty, noisy chewing, open mouth chewing, pain during the chewing, difficulty swallowing and cracking during chewing. A questionnaire was administered consisting of sociodemographic variables for the following information: I) sex (male/female); II) age (in years), categorized into 18-30 years, 31-60 years or 61 years or older; and III) education (in full years), categorized in 0-9 years, 10-12 years and 12 years or more. Additionally, independent variables, which had response options of ‘‘yes’’, ‘‘no’’, or ‘‘no response’’, included

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Table 1 - Association between adequate chewing and the associated factors. Porto Alegre, Brazil, 2012. Variables Sex Male Female Age In years Education (years) 0-9 10-12 o12 Tooth loss No Yes Use of dental prosthesis No Yes Keep their mouth open most of the time No Yes Pre-treatment of speech-language pathology No Yes

Inadequate chewing

Adequate chewing

p-value

17 (32.7%) 35 (67.3%)

370 (42.4%) 502 (57.6%)

0.107

67.2 (± 16.5)

47.8 (± 19.3)

o0.001*

7 (15.9%) 12 (27.3%) 25 (56.8%)

90 (12.5%) 193 (26.7%) 439 (60.8%)

0.778

18 (34.6%) 34 (65.4%)

644 (73.9%) 228 (26.1%)

o0.001

20 (38.5%) 32 (61.5%)

682 (78.4%) 188 (21.6%)

o0.001

45 (86.5%) 7 (13.5%)

836 (96.3%) 32 (3.7%)

o0.001

51 (98.1%) 1 (1.9%)

871 (99.8%) 2 (0.2%)

0.160**

Chi-square test (no symbol). * T-Test for independent samples. ** Fisher’s exact test.

Figure 1 - Assessment of age by (A) chewing ability and (B) number of chewing disorder alterations. Porto Alegre, Brazil, 2012.

’ DISCUSSION

chewing ranged from 47.5% to 51.8% (3). In contrast to the findings of this study, the prevalence ranged from 5.2% in adults to 11.8% in the elderly. Some of these differences can be explained by the fact that each study used different methodologies, either in terms of sampling or in the detection of chewing alteration, from self reports to evaluation by an examiner with different degrees of training, which affect the ability to compare differences in prevalence. In the process of ageing, the stomatognathic system undergoes several physiological changes.These changes may be both neurological and anatomical and may result in decreased neuromuscular activity, reflexes, sensitivity, saliva production, sense of taste, strength and tongue movements. It is also known that with advanced age, masticatory work is less efficient, and the strength employed is lower (18), which may hamper the physiological act of chewing. However, the oral health of the elderly also has a great influence, being strongly related to the presence of cavities, periodontitis, xerostomia, tooth loss and/or unadjusted prostheses (19-21). All these

In this study, it was possible to evaluate self-reported chewing alterations of a sample from Porto Alegre, southern Brazil. It was observed that inadequate chewing was found in only 5.6% of the interviewed population. To our knowledge, this is the first study to assess adults and elderly individuals based on data from a population-based survey and a probabilistic sample stratified by multiple stages. It was found that adult and older individuals who wear unadjusted prostheses prefer foods with a softer consistency, have difficulty breathing through the nose and have a higher prevalence of experiencing inadequate chewing. Few studies have investigated the prevalence of chewing alterations in adult populations. Among individuals aged between 20 and 59 years in Florianópolis (Brazil), the chewing difficulty was 13% for men and 18% for women (17). In a study of elderly individuals aged 65 to 74 years old in 250 cities in all Brazilian states, the prevalence of unsatisfactory

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Table 2 - Poisson regression with robust variance of adequate chewing in adults and the elderly. PR=prevalence ratio. Porto Alegre, Brazil, 2012.

Age (years) 18-30 31-60 61 or more Sex Male Female Education (years) 0-9 10-12 o12 Oral Health Has all teeth Lost teeth and does not use prosthesis Lost teeth and uses adjusted prosthesis Lost teeth and uses unadjusted prosthesis Preference for foods of soft consistency No Yes Difficulty breathing No Yes

Crude PR (95% CI)

Model 1a PR (95% CI)

185 (24.6) 334 (44.5) 232 (30.9)

1 7.70 (1.02-58.14) 27.08 (3.74-195.84)

1 6.05 (0.79-46.45) 22.56 (3.16-161.12)

1 4.19 (0.54-32.34) 9.41 (1.25-70.55)

1 4.22 (0.54-32.68) 9.03 (1.20-67.91)

0.168 0.032

320 (42.6) 431 (57.4)

1 1.48 (0.84-2.61)

1 1.18 (0.64 - 2.19)

1 1.01 (0.58-1.77)

1 1.15 (0.65-2.03)

0.627

93 (12.4) 200 (26.6) 458 (61.0)

1 0.81 (0.33-1.99) 0.75 (0.33-1.68)

1 0.92 (0.38-2.23) 0.97 (0.44-2.11)

1 0.87 (0.38-1.98) 1.06 (0.51-2.21)

1 0.62 (0.24-1.62) 1.12 (0.59-2.24)

0.329 0.753

1 2.58 (0.90-7.37) 2.81 (1.39-5.71) 16.71 (9.27-30.13)

1 1.49 (0.52-4.20) 1.71 (0.83-3.52) 3.71 (1.68-8.18)

1 1.56 (0.56-4.35) 1.99 (0.99-4.00) 3.50 (1.54-7.95)

0.397 0.054 0.003

738 (98.1) 14 (1.9)

1 26.68 (19.18-37.10)

1 10.15 (5.37-19.16)

1 9.34 (4.66-18.70)

670 (89.2) 81 (10.8)

1 3.51 (1.70-7.28)

553 46 123 30

(73.6) (6.1) (16.2) (4.0)

Model 2b PR (95% CI)

Model 3c PR (95% CI)

p-valued

Variables

1 2.82 (1.31-6.06)

o0.001

0.008

Adjusted for age, sex and education. Adjusted for model 1, oral health and preference for soft consistency food. c Adjusted for model 2 and difficulty breathing through the nose. d p-value model 3. b

similar characteristics and habits in terms of eating and cultural habits. In addition to the consistency of the findings with those in the literature, the prevalence and associations presented in this study are relevant for the planning of health policies aimed at promoting integral healthcare for adults and the elderly. This study has some limitations. Despite its random sample of individuals at multiple stages of life, women were the main respondents and constituted the majority of the sample, although it is well known that men die more frequently in all age groups than women. However, this factor was corrected in the multivariate analysis. Additionally, a variation in the outcome measure could be expected from the self-report survey. However, the literature is already consistent in stating that for chronic diseases, selfreports are usually reliable (30-32). Age, oral health status through dental prosthesis, preference for foods of soft consistency and difficulty breathing through the nose are factors associated with masticatory inadequacy in adults and the elderly. Thus, it is extremely important for healthcare teams to carefully investigate and evaluate these factors.

changes, whether associated or not, contribute to inadequate chewing (18,21). The findings of this study indicate that the effect of age had a major influence on the masticatory inadequacy of individuals older than 60 years. Among the main results presented, the use of total dental prosthesis negatively interferes with chewing and food preference for softer consistency. The total number of teeth is directly related to chewing ability, thus influencing food choices (22,23). The more teeth, the greater the possibility of choosing foods that are not soft or that are difficult to chew (24). Individuals with masticatory difficulties tend to consume less fibre, thus becoming more vulnerable to nutritional deficiencies and gastrointestinal diseases (25). In contrast, tooth loss and masticatory difficulty favour the selection of more easily crushed foods, which generally have fewer essential nutrients, including protein, fibre, vitamin D, niacin, pantothenic acid, vitamins B1 and B6 (24,26). In addition, eating habits, especially those related to the consistency of the food ingested, have not been evaluated in studies of chewing ability (3,7,17). It is well known that there is interference between breathing and chewing, especially when both functions compete with each other (27,28), and this interference becomes even more relevant in colder regions, such as southern Brazil, where people have more respiratory complaints. Studies also show that oral breathing interferes negatively with food leftovers in the oral cavity, lip positioning and noise while chewing (18), although there is no proven association with nutritional status (29). Faced with greater scientific evidence on these changes, the planning of health activities in the early stages of pathological development prior to main clinical manifestations is critical for greater control of these alterations, which strongly impact public health. This is an original population-based study with data that represent the southern Brazilian population, which has

â&#x20AC;&#x2122; AUTHOR CONTRIBUTIONS Baumgarten A, Schmidt JG and Rech RS were responsible for the data analysis, interpretation of results, drafting of the manuscript and critical review of the manuscript. Hilgert JB was responsible for the coordination and critical review of the manuscript. de Goulart BN was responsible for the conception and design of the study, coordination, data collection, data analysis, interpretation of results and critical review of the manuscript.

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2. Horie T, Kanazawa M, Komagamine Y, Hama Y, Minakuchi S. Association between near occlusal contact areas and mixing ability. J Oral Rehabil. 2014;41(11):829-35, http://dx.doi.org/10.1111/joor.12216. 3. Dias-da-Costa JS, Galli R, Oliveira EA, Backes V, Vial EA, Canuto R, et al. Prevalência de capacidade mastigatória insatisfatória e fatores associados em idosos brasileiros. Cad Saude Publica. 2010;26(1):79-88, http://dx.doi.org/10.1590/S0102-311X2010000100009. 4. Rodrigues HL Jr, Scelza MFZ, Boaventura GT, Custódio SM, Moreira EA, Oliveira Dde L. Relation between oral health and nutritional condition in the elderly. J Appl Oral Sci. 2012;20(1):38-44, http://dx.doi.org/10.1590/ S1678-77572012000100008. ́ 5. Sierpin´ska T, Go"ebiewska M, D"ugosz JW. The relationship between masticatory efficiency and the state of dentition at patients with non rehabilitated partial lost of teeth. Adv Med Sci. 2006; 51 Suppl 1:196-9. 6. Kumar Y, Chand P, Arora V, Singh S V., Mishra N, Alvi HA, et al. Comparison of rehabilitating missing mandibular first molars with implant- or tooth-supported prostheses using masticatory efficiency and patient satisfaction outcomes. J Prosthodont. 2017;26(5):376-80, http://dx. doi.org/10.1111/jopr.12399. 7. Jorge TM, Bassi AK, Yarid SD, Silva HM, Silva RP, Caldana ML, et al. Relação entre perdas dentárias e queixas de mastigação, deglutição e fala em indivíduos adultos. Rev CEFAC. 2009; 11 (Suppl 3):391-7. 8. Kapur KK, Soman SD. Masticatory performance and efficiency in denture wearers. 1964. J Prosthet Dent. 2006;95(6):407-11, http://dx.doi.org/10.1016/ j.prosdent.2006.03.012. 9. Hsu HY, Yamaguchi K. Decreased chewing activity during mouth breathing. J Oral Rehabil. 2012;39(8):559-67, http://dx.doi.org/10.1111/ j.1365-2842.2012.02306.x. 10. Masumoto N, Yamaguchi K, Fujimoto S. Daily chewing gum exercise for stabilizing the vertical occlusion. J Oral Rehabil. 2009;36(12):857-63, http://dx.doi.org/10.1111/j.1365-2842.2009.02010.x. 11. Nagaiwa M, Gunjigake K, Yamaguchi K. The effect of mouth breathing on chewing efficiency. Angle Orthod. 2016;86(2):227-34, http://dx.doi.org/ 10.2319/020115-80.1. 12. Rossi RC, Rossi NJ, Rossi NJ, Yamashita HK, Pignatari SSN. Dentofacial characteristics of oral breathers in different ages: a retrospective case-control study. Prog Orthod. 2015;16:23, http://dx.doi.org/10.1186/s40510-015-0092-y. 13. Lo YT, Wahlqvist ML, Chang YH, Lee MS. Combined effects of chewing ability and dietary diversity on medical service use and expenditures. J Am Geriatr Soc. 2016;64(6):1187-94, http://dx.doi.org/10.1111/jgs.14150. 14. Alfonsi E, Cosentino G, Mainardi L, Schindler A, Fresia M, Brighina F, et al. Electrophysiological investigations of shape and reproducibility of oropharyngeal swallowing: interaction with bolus volume and age. Dysphagia. 2015;30(5):540-50, http://dx.doi.org/10.1007/s00455-015-9634-1. 15. Nascimento WV, Santos CM, Cassiani RA, Dantas RO. Influence of age on swallows of a highly viscous liquid bolus. Arq Gastroenterol. 2015; 52(1):32-6, http://dx.doi.org/10.1590/S0004-28032015000100008. 16. Goulart BN, Martins-Reis VO, Chiari BM. Household survey on self-declared communication disorders: study design and protocol. Audiol Commun Res. 2015;20(4):336-48, http://dx.doi.org/10.1590/2317-6431-2015-1586. 17. Figueiredo Dde R, Peres MA, Luchi CA, Peres KG. Fatores associados as dificuldades de adultos na mastigacao. Rev Saude Publ. 2013;47(6):102838, http://dx.doi.org/10.1590/S0034-89102013000901028.

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CLINICAL SCIENCE

Prognostic factors of the short-term outcomes of patients with hepatitis B virus-associated acute-on-chronic liver failure Qing Lei,I,# Kangjian Ao,I,# Yinhua Zhang,I Deqiang Ma,I Deping Ding,I Changzheng Ke,I Yue Chen,I Jie Luo,III,* Zhongji MengI,II,* I Department of Infectious Diseases, Taihe Hospital, Hubei University of Medicine, Hubei, Shiyan, 442000, China. II Institute of Biomedical Research, Taihe Hospital, Hubei University of Medicine, Hubei, Shiyan, 442000, China. III Center for Evidence-Based Medicine and Clinical Research, Taihe Hospital, Hubei University of Medicine, Hubei, Shiyan, 442000, China.

OBJECTIVE: To investigate the impact of the baseline status of patients with hepatitis B virus-associated acuteon-chronic liver failure on short-term outcomes. METHODS: A retrospective study was conducted that included a total of 138 patients with hepatitis B virusassociated acute-on-chronic liver failure admitted to the Department of Infectious Diseases, Taihe Hospital, Hubei University of Medicine, from November 2013 to October 2016. The patients were divided into a poor prognosis group (74 patients) and a good prognosis group (64 patients) based on the disease outcome. General information, clinical indicators and prognostic scores of the patients’ baseline status were analyzed, and a prediction model was established accordingly. RESULTS: Elder age, treatment with artificial liver support systems and the frequency of such treatments, high levels of white blood cells, neutrophils, neutrophil count/lymphocyte count ratio, alanine aminotransferase, gamma-glutamyl transferase, total bilirubin, urea, and prognostic scores as well as low levels of albumin and sodium were all significantly associated with the short-term outcomes of hepatitis B virus-associated acute-onchronic liver failure. The predictive model showed that logit (p) = 3.068 + 1.003 neutrophil count/lymphocyte count ratio - 0.892 gamma-glutamyl transferase - 1.138 albumin - 1.364 sodium + 1.651 artificial liver support therapy. CONCLUSION: The neutrophil count/lymphocyte count ratio and serum levels of gamma-glutamyl transferase, albumin and sodium were independent risk factors predicting short-term outcomes of hepatitis B virusassociated acute-on-chronic liver failure, and the administration of multiple treatments with artificial liver support therapy during the early stage is conducive to improved short-term outcomes. KEYWORDS: Hepatitis B Virus; Acute-on-Chronic Liver Failure; Prognosis. Lei Q, Ao K, Zhang Y, Ma D, Ding D, Ke C, et al. Prognostic factors of the short-term outcomes of patients with hepatitis B virus-associated acuteon-chronic liver failure. Clinics. 2017;72(11):686-692 Received for publication on March 20, 2017; First review completed on June 19, 2017; Accepted for publication on August 14, 2017 *Corresponding author. E-mail: zhongji.meng@163.com / jie.luo@aliyun.com #

These authors contributed equally to this work.

’ INTRODUCTION

common type of liver failure in China and has a high mortality rate of 50-90% (1). The predominant causes of ACLF differ in different parts of the world. In China, the most important cause is infection with hepatitis virus, especially hepatitis B virus (HBV) (2). The pathogenesis of HBV-ACLF has not been well elucidated, although the ‘‘three-shock’’ hypothesis (i.e., immune injury, ischemia and hypoxia and endotoxin-induced damage) is currently the most generally recognized hypothesis (3). For severe ACLF, the only effective treatment is liver transplantation. However, due to the shortage of resources, the demand for liver transplantation cannot be met. At present, the treatment strategy for HBV-ACLF is comprehensive combination with internal medicine, anti-viral medications and artificial liver support therapy (4). Artificial liver support

Acute-on-chronic liver failure (ACLF) refers to a group of complex clinical syndromes characterized by acute severe liver function damage (caused by a number of acute triggering factors) in patients with chronic liver disease, complicated by the failure of one or more organs. ACLF is the most

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Inclusion and exclusion criteria

therapy can temporarily replace part of the liver function and prevent further exacerbation of liver failure by removing toxic substances and metabolites from the serum, improving the microenvironment for liver cell regeneration and liver function repair. However, the efficacy of artificial liver support therapy for liver failure remains controversial (5-7). The early identification, accurate diagnosis and prognostic evaluation of ACLF can provide a guiding basis for active and effective treatment. Therefore, a better understanding of prognostic factors and more precise prognostic evaluation systems for ACLF are in urgent need. A variety of factors can affect the progression and prognosis of ACLF. Many prognostic scoring systems are available for predicting the outcomes of ACLF, including the Child-Turcotte-Pugh (CTP) system, the model for end-stage liver disease (MELD) and the MELD-sodium (MELD-Na). Each scoring system has certain limitations given that not all of the influencing factors can be included in the individual assessment. The integrated MELD (iMELD) is a new scoring system that features the addition of two independent ACLF prognostic risk factors, age and serum Na levels, to the MELD scoring system (8). In addition, the albumin-bilirubin (ALBI) grading system is a recently developed scoring system to assess liver function (9). Several studies have compared the predictive ability of different scoring systems in ACLF (10-12). Comparisons of the predictive abilities of the CTP, MELD, MELD-Na, iMELD and ALBI scoring systems for the prognosis of HBV-ACLF are rarely reported. Attempts to analyze the impact of clinical parameters and the combined prognostic abilities of these parameters on ACLF prognosis have provided inconsistent results due to differences in study subjects, study phases and follow-up periods (13-15). The integration of general information, clinical indicators and a prognostic scoring system may better predict the short-term outcomes of patients with HBV-ACLF. In this study, a retrospective investigation was carried out in HBV-ACLF patients admitted to the Department of Infectious Diseases, Taihe Hospital, Hubei University of Medicine from November 2013 to October 2016. General patient information, laboratory indicators and prognostic scores at baseline of HBV-ACLF patients with different prognoses were analyzed, and the factors that influenced the short-term outcomes of HBV-ACLF were investigated.

The diagnostic criteria for HBV-ACLF were based on the ‘‘Guidelines for the Prevention and Treatment of Chronic Hepatitis B’’ of China issued in 2015 and the ‘‘Guidelines for Diagnosis and Treatment of Liver Failure (2012 Edition)’’ of China. The exclusion criteria included patients complicated with other forms of viral hepatitis, alcoholic liver disease, autoimmune liver disease, drug-induced liver injury, associated tumors or severe organ disease other than hepatitis B.

General information The general information included gender, age, with/ without (w/wo) liver cirrhosis, rebound after withdrawal of anti-viral drugs, complication with ascites at admission and the acceptance and frequency of artificial liver support therapy. All of this information was retrieved from the medical record system of Taihe Hospital.

Clinical indicators The following measurements were performed using venous blood collected on the first day of admission or on the morning following admission: baseline white blood cell count (WBC), absolute neutrophil count (NE), absolute lymphocyte count (LY), NE:LY ratio (NE/LY), platelet count (PLT), levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyltransferase (GGT), albumin (Alb), total bilirubin (TBil), urea (Urea), and creatinine (Cr), prothrombin time (PT), prothrombin activity (PTA), activated partial thromboplastin time (APTT), the international normalized ratio (INR) and serum Na levels. All results were retrieved from the clinical database.

Prognostic score The prognostic scoring systems included the CTP, MELD, MELD-Na, iMELD and ALBI. The CTP score is the cumulative result of the scores for five items (ascites, hepatic encephalopathy, TBil, Alb and PT extension time), with 1-3 points for each item and a maximum of 15 points (16, 17). The equation for the MELD score is as follows: 3.8 LN (TBil [mg/dL]) + 11.2 LN (INR) + 9.6 LN (Cr [mg/dL]) + 6.4 cause (0 for cholestatic or alcoholic liver diseases and 1 for all others); the result is a rounded integer (18). The equation for the MELD-Na score is as follows: MELD + 1.59 (135 - Na), wherein Na is 135 mmol/L if Na 4 135 mmol/L and 120 mmol/L if Na o 120 mmol/L (19). The equation for the iMELD score is as follows: MELD + (0.3 Age) - (0.7 Na) + 100 (8). The equation for the ALBI score is as follows: (log10TBil [mmol/L] 0.66) + (Alb [g/L] - 0.085) (9).

’ PATIENTS AND METHODS Patient selection Based on the clinical data in the medical record system of Taihe Hospital, Hubei University of Medicine, a retrospective analysis was conducted in patients admitted to the Department of Infectious Diseases from November 2013 to October 2016 who met the criteria of HBV-ACLF during their hospitalization. The data from the medical records of the selected patients were input in the form of case reports and verified with the clinical data system in our hospital. All of the patients were given comprehensive supportive treatment of internal medicine after admission to the hospital, including anti-viral therapy with nucleoside analogs. Artificial liver support therapy was optional based on the patient’s condition and willingness. The end point of the observation in this study was the time of discharge or in-hospital death of the patient. The study protocol was approved by the Ethics Committee of Taihe Hospital, Hubei University of Medicine.

Prognostic criteria The prognostic criteria in this study were based on the ACLF clinical improvement criteria in ‘‘Guidelines for Diagnosis and Treatment of Liver Failure (2012 Edition)’’ of China (2). A good prognosis needs to meet all of the following conditions simultaneously: 1) the clinical symptoms are significantly improved and hepatic encephalopathy has disappeared; 2) signs of jaundice and ascites are significantly improved; and 3) liver function is significantly improved (TBil o5 ULN, PTA 440%). The patients who died, whose condition became more advanced, and those who still experienced ACLF were included in the poor prognosis group.

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Figure 1 - Case screening and enrollment. No statistically significant difference was observed between the poor prognosis group and the good prognosis group in terms of disease stage at admission.

Table 1 - General information of the patients.

Age (years) Gender (male/female) W/wo liver cirrhosis W/wo drug-withdrawal rebound W/wo ascites W/wo ALST Number of treatments

Good prognosis

Poor prognosis

Statistics value

p value

43.16±12.44 52/12 19/45 6/5 41/23 54/10 1.62 (0.79, 2.55)

48.08±9.08 59/15 32/42 10/22 55/19 36/38 0.64 (0, 1.61)

t0 = 2.621 w2 = 0.05 w2 = 2.707 w2 = 1.035 w2 = 1.707 w2 = 19.31 Z = -4.422

0.010 0.822 0.100 0.309 0.191 0.000 0.000

Note: W/wo indicates with/without; ALST is the abbreviation of artificial liver support therapy.

’ RESULTS

Data processing All data included in this study were retrieved from the electronic medical record system of Taihe Hospital and validated using the clinical data system. Cases with missing or incomplete data, such as AFP data, were not included in the study.

Case enrollment A total of 171 patients with HBV-ACLF were admitted to the department of infectious disease of Taihe Hospital from November 2013 to October 2016. A total of 138 patients were selected according to the inclusion and exclusion criteria, including 111 males and 27 females with an average age of 45.80± 11.01 years. There were 74 cases in the poor prognosis group, accounting for 53.6%, and 64 cases in the good prognosis group, accounting for 46.4%. Of the cases in the poor prognosis group, 59 cases showed ACLF; 15 cases were in the pre-ACLF state at admission. In the good prognosis group, the corresponding numbers were 45 cases and 19 cases, respectively (Figure 1).

Statistical analysis Statistical Package for the Social Sciences (SPSS) 17.0 software was used for the statistical analysis. The measurement data were first tested for a normal distribution. Data that showed a normal distribution were represented as x–±s and were analyzed using the t-test. The data that were not normally distributed were represented as medians (P25 and P75) and analyzed using the nonparametric rank sum test. The count data among different groups were analyzed using the w2 test. The area under the receiver operating characteristic (ROC) curve was used to assess the predictive power of the five scoring systems for the prognosis of HBV-ACLF, and the cut-off value of the continuous variable was calculated. The method of likelihood-ratio-forward-selection in nonconditional binary logistic regression analysis was used to obtain the independent risk factors and to establish a predictive model. The threshold used for statistical significance was po0.05.

General information of the patients The average age of the patients in the poor prognosis group was significantly higher than that in the good prognosis group (48.08±9.08 years vs. 43.16±12.44 years, p=0.01). The number of patients who received artificial liver support therapy and the frequencies of artificial liver support therapy in the good prognosis group were significantly higher than in the poor prognosis group (po0.01). There were no significant differences between the two groups with respect to sex, rebound after anti-viral drug withdrawal or the complications of cirrhosis and ascites (Table 1).

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Table 2 - Clinical indicators of the baseline status of the patients.

WBC ( 10 /L) NE ( 1012/L) LY ( 1012/L) NE/LY PLT ( 1012/L) ALT (U/L) AST (U/L) GGT (U/L) Alb (g/L) TBil (mmol/L) Urea (mmol/L) Cr (mmol/L) Na (mmol/L) PT (s) PTA (%) APTT (s) INR 12

Good prognosis

Poor prognosis

Statistics value

p value

5.31 (4.12, 6.79) 3.16 (2.28, 4.99) 1.13 (0.84, 1.43) 2.70 (1.79, 5.37) 88.0 (65.25, 125.67) 507.5 (175.5, 902.0) 289.5 (124.0, 767.5) 108.5 (62.5, 159.5) 33.74±5.174 234.24±106.27 4.17 (2.98, 5.44) 55.75 (42.7, 77.95) 138.91±4.209 22.45 (18.5, 26.03) 28.27 (22.64, 36.97) 52.83±14.70 1.98±0.62

6.40 (4.48, 9.07) 4.03 (2.86, 6.57) 1.21±0.63 3.96 (2.93, 6.69) 85.0 (46.0, 122.33) 198.0 (81.0, 674.0) 210.0 (95.67, 424.0) 66.0 (47.0, 125.0) 30.71±5.56 301.62±144.45 4.54 (3.54, 7.52) 56.95 (42.7, 85.5) 136.07±6.33 25.0 (18.6, 30.9) 26.37±12.80 56.33±18.69 2.05 (1.6, 2.72)

Z = -2.291 Z = -2.248 Z = -0.566 Z = -2.517 Z = -1.087 Z = -2.589 Z = -1.543 Z = -2.735 t = -3.30 t0 = 3.147 Z = -2.002 Z = -0.431 t0 = -3.136 Z = -1.738 Z = -1.964 t0 = 1.231 Z = -1.642

0.022 0.025 0.572 0.012 0.277 0.01 0.123 0.006 0.001 0.002 0.045 0.666 0.002 0.082 0.05 0.22 0.101

Note: Normally distributed data are represented as x–±s. Non-normally distributed data are represented as medians (P25 and P75).

Table 3 - Prognostic scores of the patients at baseline.

CTP score MELD score MELD-Na score iMELD score ALBI score

Good prognosis

Poor prognosis

10.11±1.78 18.08±7.10 18.95±7.7 33.79±9.15 -1.34±0.52

11.2±1.68 21.58±7.39 24.18±10.11 40.75±9.90 -1.01±0.54

p value

Statistics value t t t t t

= = = = =

3.706 2.828 3.573 4.268 3.639

0.000 0.005 0.000 0.000 0.000

Table 4 - Comparison of the five types of prognostic scores. Area

CTP MELD MELD-Na iMELD ALBI Logit(p)

0.672 0.641 0.656 0.699 0.682 0.656

Sensitivity (%)

Specificity (%)

47.3 56.8 41.9 74.3 62.2 62.2

79.7 71.9 85.9 62.5 67.2 64.1

Std. error

0.045 0.047 0.046 0.045 0.045 0.046

The baseline clinical indicators for HBV-ACLF patients with different prognoses

Asymptotic Sig.

0.000 0.004 0.002 0.000 0.000 0.002

95% C.I. Lower

Upper

0.593 0.549 0.565 0.611 0.594 0.565

0.761 0.733 0.746 0.786 0.771 0.746

prognostic scores. The areas under the ROC curves of the CTP, MELD, MELD-Na, iMELD and ALBI prognostic systems were 0.672, 0.641, 0.656, 0.699, and 0.682, respectively (Table 4, Figure 2).

The WBC, NE, NE/LY, ALT, GGT, TBil and Urea scores/ levels were significantly higher in the poor prognosis group (po0.05), while the Alb and Na levels in the good prognosis group were significantly higher than in the poor prognosis group (po0.01). There were no statistically significant differences between the two groups in terms of the other clinical indicators, including LY, PLT, AST, Cr, PT, APTT and INR (Table 2).

Independent prognostic factors and novel prediction model for HBV-ACLF The different measures of general patient information, clinical indicators and prognostic scores at baseline for the two groups were assigned corresponding values for nonconditional binary logistic regression analysis. The cut-off values of the continuous variables were calculated using the area under the ROC curve (Tables 5 and 6) to establish the following prediction model: logit (p) = 3.068 + 1.003 NE/ LY - 0.892 GGT - 1.138 Alb -1.364 Na + 1.651 artificial liver support therapy. The area under the ROC curve was 0.656, with a specificity of 64.1% and sensitivity of 62.2%. NE/LY, GGT, Alb, Na and artificial liver support therapy were the independent factors influencing the shortterm outcomes of HBV-ACLF (Table 7).

High CTP, MELD, MELD-Na, iMELD and ALBI scores all predict poor short-term outcomes for HBV-ACLF patients Compared with the patients with a good prognosis, the patients with a poor prognosis showed significantly higher scores for the CTP, MELD, MELD-Na, iMELD and ALBI prognostic systems (po0.05) (Table 3). The prognosis of HBV-ACLF was well predicted by these five types of

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Table 6 - Variable assignment. Assignment

Age WBC NE NE/LY ALT GGT Alb TBil Urea Na MELD MELD-Na iMELD CTP ALBI Artificial liver support therapy (ALST) Prognosis

Table 5 - Area under the ROC curve and the cut-off value of each variable.

Age WBC NE NE/LY ALT GGT Alb TBil Urea Na CTP MELD MELD-Na iMELD ALBI

43.5 6.75 5.635 2.947 127.5 76 34.85 251.2 6.365 136.3 11.5 21.448 25.636 34.705 -1.119

c-statistic

0.643 0.613 0.611 0.624 0.372 0.365 0.343 0.632 0.599 0.361 0.672 0.641 0.656 0.699 0.682

95% CI Lower

Upper

0.549 0.52 0.517 0.53 0.279 0.272 0.252 0.54 0.505 0.268 0.593 0.549 0.565 0.611 0.594

0.738 0.707 0.705 0.719 0.465 0.457 0.435 0.725 0.693 0.454 0.761 0.733 0.746 0.786 0.771

p 43.5 p 6.75 p 5.635 p 2.947 p 127.5 p 76 p 34.85 p 251.2 p 6.365 p 136.3 p 21.448 p 25.636 p 34.705 p 11.5 p -1.119 No Good prognosis

4 43.5 4 6.75 4 5.635 4 2.947 4 127.5 4 76 4 34.85 4 251.2 4 6.365 4 136.3 4 21.448 4 25.636 4 34.705 4 11.5 4 -1.119 Yes Poor prognosis

artificial liver therapy can improve the conditions and opportunities for the regeneration of liver cells and the recovery of liver function. Artificial liver support systems take various forms, and their efficacy in treating liver failure remains controversial. Previous studies have shown that artificial liver support therapy can only improve the bilirubin level and hepatic encephalopathy of the patients, with no effect on the survival rate (5). There are also studies indicating that artificial liver support therapy can reduce the short-term mortality rate of patients with ACLF, whereas its impact on long-term survival is uncertain (6). Considering that the above results are for different types of artificial liver support therapy and different periods of application, plasma replacement therapy was primarily applied in this study. The number of patients who received artificial liver support therapy and the frequency of this therapy were significantly higher in the good prognosis group than in the poor prognosis group. Moreover, artificial liver support therapy was an independent factor for the short-term outcomes of HBV-ACLF. Therefore, the early active implementation of treatment combined with artificial liver support therapy can significantly improve the short-term outcomes of patients with HBV-ACLF. WBC, NE and the NE/LY ratio are often associated with inflammation and infection. According to the ‘‘three-shock’’ hypothesis, inflammation is an important step in the development of HBV-ACLF (3). At the same time, studies have shown that infection can induce and promote the development of ACLF (22), and the severity of inflammation is significantly correlated with the prognosis of ACLF (23) The study of Li et al (13) showed that WBC is an independent risk factor for HBV-ACLF. In this study, the WBC, NE count and NE/LY ratio of the patients with a poor prognosis were significantly higher than those of the patients with a good prognosis, suggesting that, in addition to WBC, a high NE count and NE/LY ratio are also important prognostic factors for HBV-ACLF. The following events occur in cases of liver failure: liver cell necrosis occurs; liver function is impaired; enzyme synthesis is reduced; disorders in detoxification, excretion and transport capacity occur; bilirubin, amino acids and other substances are released into the blood and accumulate; serum bilirubin and transaminase levels increase; and the

Figure 2 - ROC curves for CTP, MELD, MELD-Na, iMELD, ALBI score and logit (p) curve.

Cut-off value

’ DISCUSSION HBV-ACLF is a common fatal clinical disease characterized by a large number of necrotic liver cells, a complex pathological mechanism and rapid progression. The results of this study showed that many factors are closely related to the short-term outcomes of this condition. Age has been shown to be closely related to the severity of liver diseases and serves as an independent prognostic factor for end-stage liver disease (8, 20). Studies have found that the short-term mortality rate of patients with ACLF is positively correlated with patient age (21). In this study, the average age of the patients with a poor prognosis was significantly higher than that in the patients with a good prognosis, suggesting that age is an important prognostic factor in HBV-ACLF. As an important supportive treatment measure for the recovery of liver function in patients with liver failure,

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Table 7 - Logistic regression analysis results. B

NE/LY GGT Alb Na Artificial liver support therapy Constant term

1.003 -0.892 -1.138 -1.364 1.651 3.068

0.444 0.428 0.511 0.455 0.473 1.441

Wald

5.109 4.338 4.951 8.979 12.161 4.535

Sig

0.024 0.037 0.026 0.003 0.000 0.033

Exp(B)

2.728 0.41 0.321 0.256 5.21 21.489

95% CI for Exp(B) Lower

Upper

1.143 0.177 0.118 0.105 2.06

6.512 0.949 0.873 0.624 13.174

higher than those of the patients in the good prognosis group, suggesting that the prognosis of HBV-ACLF was significantly correlated with the prognostic scores at baseline status. A novel prognostic scoring system was established according to the factors that affect the short-term outcomes of HBVACLF, as follows: logit (p) = 3.068 + 1.003 NE/LY - 0.892 GGT - 1.138 Alb - 1.364 Na + 1.651 artificial liver support therapy. The sensitivity and specificity were 62.2% and 64.1%, respectively. The sensitivity of this novel predictive model was much higher than those of MELD-Na and MELD scores. Additionally, the area under the ROC curve of this predictive model was much larger than that of the MELD score, although it was smaller than that of iMELD score. Most importantly, except for serum Na, the parameters of NE/LY, GGT, Alb, and artificial liver support therapy were shown to be important factors that affect the short-term outcomes of HBV-ACLF as well. In addition to the indexes of liver function, complications such as infection were also included in this model. Thus, the novel predictive model was extremely suitable for ACLF patients in the end stage with multiple complications, especially those with an extremelyelevated INR, and this circumstance was restricted in evaluations using the iMELD, MELD-Na, and MELD scores. Differences in study subjects, study phases and sample sizes may lead to different results. Although this study was a comprehensive study of factors that contribute to the shortterm outcomes of HBV-ACLF, it had some limitations. First, all patients were enrolled from a single center, which may not reflect regional influences on the prognosis. Secondly, alpha fetal protein and a history of antiviral therapy were not examined in this study. Lastly, only short-term outcomes were analyzed. Multi-center studies over different observation periods are required to further research HBV-ACLF and to provide a better theoretical basis for its clinical diagnosis and treatment. In conclusion, a variety of factors can affect the prognosis of HBV-ACLF patients. Older age, high WBC, NE, NE/LY, and TBil levels, renal dysfunction, hyponatremia, hypoproteinemia and high prognostic scores at baseline often suggest a poor prognosis. For patients with HBV-ACLF, within the context of anti-viral treatment and comprehensive internal medicine treatment, infection control should be strengthened and the stability of the patient’s lab values, including the electrolyte balance, should be maintained. In addition, early active treatment combined with artificial liver support therapy is recommended. Further studies are needed to enhance our understanding of HBV-ACLF pathogenesis and reduce its morbidity and mortality.

synthesis of proteins and coagulation factors decreases, resulting in decreased Alb levels and the development of coagulation disorders. In the present study, liver function and coagulation function were abnormal in both groups, and the levels of ALT, GGT, TBil and Alb all showed significant differences between the two groups. Of these measures, GGT and Alb were independent risk factors for poor prognosis of HBV-ACLF. However, a comparison of the baseline PTA of the patients in the two groups showed a significance level of p=0.05. Therefore, this difference was not considered statistically significant. Baseline PTA may be related to the patient’s stage of disease at admission. Because the majority of patients in this study had reached the ACLF state at admission, with PTA p40%, the difference between the two groups was small. Changes and differences in coagulation can be further investigated and analyzed at different times and over different observation periods. Ascites, hyponatremia and hepatorenal syndrome are common complications of ACLF. Complications such as refractory ascites, hyponatremia and renal injury are often interrelated and may continuously degenerate (2). In this study, the serum Na levels of patients in the poor prognosis group were significantly lower than those of the patients with a good prognosis. Low Na levels were an independent risk factor for poor prognosis of HBV-ACLF; this finding is consistent with the results of the studies of Zhang et al (15) and Shi et al (24). The results suggest that electrolytes should be closely monitored and electrolyte imbalances should be corrected during HBV-ACLF treatment. In this study, the serum levels of urea were higher in the poor prognosis group than those in patients with good prognoses, whereas no significant difference in the level of Cr was found between the two groups. Accordingly, renal function damage may have a certain relationship with the prognosis of HBV-ACLF. However, the etiology of all patients included in this study was HBV infection. The diagnostic criteria for ACLF were based on the ‘‘Guidelines for Diagnosis and Treatment of Liver Failure (2012 Edition)’’ of China. In cases of ACLF associated with HBV infection, early organ damage is primarily manifested as liver function damage and coagulation dysfunction rather than as increased Cr levels. Use of the prognostic evaluation system can help clinicians predict disease prognosis in the early stage of the disease and assess the severity of the disease, providing guidance in the choice of active and effective treatment. However, because each prognostic scoring system has limitations, this study for the first time compared the five scoring systems (CTP, MELD, MELD-Na, iMELD and ALBI) in HBV-ACLF patients with differing prognosis. The results showed that all five prognostic scoring systems demonstrated good predictive value for ACLF. The prognostic scores of the patients at baseline status in the poor prognostic group were significantly

’ ACKNOWLEDGMENTS This work was partly supported by the National Natural Science Foundation of China (81541140), Natural Science Foundation of Hubei Province

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of China (2014CFB645), Research and Development Project of the Science and Technology Plan of Hubei Province (2011BCB030), Foundation for Innovative Research Team of Hubei University of Medicine (2014CXG05) and the Key Program for Precision Medicine of Taihe Hospital (2016JZ05).

11. 12.

’ AUTHOR CONTRIBUTIONS Lei Q, Ke C, Chen Y, Luo J and Meng Z conceived and designed the study. Lei Q, Ao K, Zhang Y, Ma D, Ding D were responsible for the data collection. Lei Q analyzed the data and wrote the manuscript. Meng Z was responsible for the manuscript ﬁnal revision.

13. 14.

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Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2014;26(8):539-43, http://dx.doi. org/10.3760/cma.j.issn.2095-4352.2014.08.003. Shen Y, Liu YM, Wang B, Zhu YG, Wang YY, Wang XL, et al. External validation and comparison of six prognostic models in a prospective cohort of HBV-ACLF in China. Ann Hepatol. 2016;15(2):236-45. Peng Y, Qi X, Tang S, Deng H, Li J, Ning Z, et al. Child-Pugh, MELD, and ALBI scores for predicting the in-hospital mortality in cirrhotic patients with acute-on-chronic liver failure. Expert Rev Gastroenterol Hepatol. 2016;10(8):971-80, http://dx.doi.org/10.1080/17474124.2016.1177788. Li C, Lyu S, Zhu B, Wan ZH, Liu WS, Guo L, et al. [Risk factors for shortterm outcome of patients with HBV-related acute-on-chronic liver failure]. Zhonghua Gan Zang Bing Za Zhi. 2016;24(3):207-13. Huang K, Hu JH, Wang HF, He WP, Chen J, Duan XZ, et al. Survival and prognostic factors in hepatitis B virus-related acute-on-chronic liver failure. World J Gastroenterol. 2011;17(29):3448-52, http://dx.doi.org/10.3748/ wjg.v17.i29.3448. Zhang Q, Guo X, Zhao S, Pang X, Wang Y, Zhang Y, et al. Prognostic performance of clinical indices and model scorings for acute-on-chronic liver failure: A study of 164 patients. Exp Ther Med. 2016;11(4):1348-54, http://dx.doi.org/10.3892/etm.2016.3037. Child CG, Turcotte JG. Surgery and portal hypertension. Major Probl Clin Surg. 1964;1:1-85. Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg. 1973;60(8):646-9, http://dx.doi.org/10.1002/bjs.1800600817. Kamath PS, Wiesner RH, Malinchoc M, Kremers W, Therneau TM, Kosberg CL, et al. A model to predict survival in patients with end-stage liver disease. Hepatology. 2001;33(2):464-70, http://dx.doi.org/10.1053/ jhep.2001.22172. Biggins SW, Kim WR, Terrault NA, Saab S, Balan V, Schiano T, et al. Evidence-based incorporation of serum sodium concentration into MELD. Gastroenterology. 2006;130(6):1652-60, http://dx.doi.org/10.1053/j.gastro. 2006.02.010. Qin G, Shao JG, Wang B, Shen Y, Zheng J, Liu XJ, et al. Artificial liver support system improves short- and long-term outcomes of patients with HBV-associated acute-on-chronic liver failure: a single-center experience. Medicine (Baltimore). 2014;93(28):e338, http://dx.doi.org/10.1097/MD. 0000000000000338. Qin G, Shao JG, Zhu YC, Xu AD, Yao JH, Wang XL, et al. Populationrepresentative Incidence of Acute-On-Chronic Liver Failure: A Prospective Cross-Sectional Study. J Clin Gastroenterol. 2016;50(8):670-5, http://dx.doi. org/10.1097/MCG.0000000000000538. Nanchal RS, Ahmad S. Infections in Liver Disease. Crit Care Clin. 2016; 32(3):411-24, http://dx.doi.org/10.1016/j.ccc.2016.03.006. Sole C, Sola E, Morales-Ruiz M, Fernandez G, Huelin P, Graupera I, et al. Characterization of Inflammatory Response in Acute-on-Chronic Liver Failure and Relationship with Prognosis. Sci Rep. 2016;6:32341, http://dx. doi.org/10.1038/srep32341. Shi KQ, Cai YJ, Lin Z, Dong JZ, Wu JM, Wang XD, et al. Development and validation of a prognostic nomogram for acute-on-chronic hepatitis B liver failure. J Gastroenterol Hepatol. 2017;32(2):497-505, http://dx.doi. org/10.1111/jgh.13502.

CLINICAL SCIENCE

Videofluoroscopic analysis of different volumes of liquid bolus swallowing in healthy individuals: comparison between height and sex Marcia Regina Kfouri Bernardi Regueiro,I Weslania Viviane Nascimento,II Luana Casari Parreira,I Roberto Oliveira DantasII,* I Departamento de Oftalmologia, Otorrinolaringologia, Cirurgia de Cabeca e Pescoco, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, BR. II Departamento de Clinica Me´dica, Faculdade de Medicina de Ribeirao Preto, Universidade de Sao Paulo, Ribeirao Preto, SP, BR.

OBJECTIVE: The volume of swallowed bolus affects the pharyngeal transit duration. The sex and corporal height of individuals may likely influence this effect. The aim of this investigation was to determine the influence of sex and corporal height on the pharyngeal transit modification produced by the swallowed bolus volume. METHODS: Forty healthy volunteers, 20 men and 20 women, including tall (10 men and 10 women, corporal height: 1.71--2.07m) and short (10 men and 10 women, corporal height: 1.52--1.70m) persons, ranging in age between 20 and 50 years, were included in the study. Videofluoroscopic evaluation of swallowing was performed with the subjects in the sitting position. Each individual swallowed three 5 mL and three 10 mL boluses of liquid barium in a random sequence. The durations of oral transit, pharyngeal transit, pharyngeal clearance, hyoid movement, upper esophageal sphincter opening and oral-pharyngeal transit were evaluated. RESULTS: In men and women, and in taller and shorter individuals, the increase of the swallowed liquid bolus volume from 5 mL to 10 mL causes a faster transit of the bolus tail from the oral-pharyngeal transition to the upper esophageal sphincter and an increase in the duration of the upper esophageal sphincter opening, with similar alteration in men and women and in taller and shorter individuals. CONCLUSION: An increase in the swallowed liquid bolus volume from 5 mL to 10 mL causes a faster pharyngeal bolus transit and a longer bolus transit through the upper esophageal sphincter, with similar alterations in men and women and in shorter and taller individuals. KEYWORDS: Deglutition; Deglutition Disorders; Swallowing; Dysphagia; Height; Sex. Regueiro MR, Nascimento WV, Parreira LC, Dantas RO. Videofluoroscopic analysis of different volumes of liquid bolus swallowing in healthy individuals: comparison between height and sex. Clinics. 2017;72(11):693-697 Received for publication on May 31, 2017; First review completed on July 13, 2017; Accepted for publication on August 31, 2017 *Corresponding author. E-mail: rodantas@fmrp.usp.br

’ INTRODUCTION

suggesting that the observed differences are likely caused by different heights of individuals, although previous results have shown a week correlation between height and some swallowing events (3). Superior hyoid displacement is greater in taller individuals than in shorter individuals (12). Height and sex have an influence on the size of the hypopharynx and the larynx, with an independent and interacting effect on the morphology of the pharynx and the larynx (13). In women the pharynx air column is surrounded by smaller structures, and there are no differences related to pharyngeal air column measurements (14). The pharyngeal cross-sectional area measured with individuals in the sitting position is greater in men than in women (15). These anatomical differences may influence the modifications caused by the swallowed bolus volume. The differences in swallowing timing in men and women, and with the swallowed bolus volume, may be important in patients with dysphagia. Some patients may not be able to swallow higher volumes because of their

The volume and consistency of the swallowed bolus have an influence on the oral and pharyngeal phases of swallowing (1-5), with an increase in upper esophageal sphincter (UES) opening duration (1,3,4) and longer pharyngeal transit (3). Several studies have demonstrated that sex can be a factor in influencing the phases of swallowing (5,6), with women having longer oral-pharyngeal transit durations than men (6-10). However, several investigations did not find that sex influences the timing of swallowing events (3,11),

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Table 1 - Age, height, weight and body mass index (BMI) of healthy individuals, men (n=20), women (n=20), taller (n=20) and shorter (n= 20) subjects, included in the investigation. MEN

Age (years) Height (m) Weight (kg) BMI (kg/m2)

WOMEN

TALLER

SHORTER

Mean

Limits

Mean

Limits

Mean

Limits

Mean

Limits

34.1 1.77 78.5 25.1

24-50 1.61-2.07 59.0-104.0 21.6-29.5

28.9 1.66 59.2 21.6

22-50 1.52-1.79 48.0-77.5 18.7-28.0

27.8 1.81 76.6 23.1

22-40 1.71-2.07 48.0-104.0 18.7-29.5

35.2 1.62 61.1 23.5

20-50 1.52-1.70 48.0-81.0 19.5-28.0

bolus tail finish to pass the UES; e) duration of hyoid movement (HM): the time interval between the onset and the end of hyoid movement; and f) oral-pharyngeal transit (OPT): the tongue tip at incisors until the bolus tail finished passing the UES (17). Statistical analysis was performed using ProEstat Estatistics and Research (Ribeirão Preto SP, Brazil) and a linear model with mixed effects (random and fixed effects) (18). In the tables, the results are shown as the mean and 95% confidence interval (95% CI), in milliseconds (ms). The individual results are shown in the figures as the mean of the three swallows performed by each individual for each volume. A pp0.05 was considered to be statistically significant.

incapacity to change the swallow dynamics and to increase the oral and pharyngeal capacity with the increase in bolus volume. Considering the hypothesis that the swallows modification caused by a 5 mL to 10 mL increase in liquid bolus volume may be different in men and women and in shorter and taller individuals, the objective of this investigation was to evaluate the modification of the oral and pharyngeal transit durations after swallows of 5mL and 10mL in men and women with different body heights.

’ MATERIAL AND METHODS Evaluation of the duration of the oral and pharyngeal phases of swallowing was performed using videofluoroscopy in 40 healthy individuals, 20 men and 20 women, ranging in age between 20 and 50 years (mean age 31.5 years) (Table 1). The height of the taller group (n=20) ranged between 1.71m and 2.07 m (mean, 1.81 m; 10 men and 10 women) and the height of the shorter group ranged between 1.52 m and 1.70 m (mean, 1.62 m; 10 men and 10 women). All volunteers had no symptoms; had no digestive, neurologic or endocrine disease; and did not undergo previous surgery. The Human Research Committee of the University Hospital of Ribeirão Preto approved the investigation (protocol HCRP 1954/2010). A written informed consent was obtained from each participant and the anonymity of each volunteer was preserved. Evaluation of swallowing was performed using the videofluoroscopy and radiologic Arcomax angiograph (model BV 300, Phillips Veenpluis, The Netherlands), which recorded 30 frames/second. The maximum duration of the tests was 60 seconds. The examination was performed with the volunteers in the lateral position seated on a chair. Non-cued swallowing of 5mL or 10mL of liquid bolus was evaluated in triplicate in a random sequence. The liquid bolus was prepared with 30mL of liquid barium sulfate (Bariogels 100%, Laboratório Cristália, Itapira SP, Brazil) diluted in 30mL of water and was given to the individual in a plastic cup with a final consistency that was classified as level 1 (slightly thick) in the gravity flow test proposed by the International Dysphagia Diet Standardisation Initiative (IDDSI) (16). The examinations were recorded for posterior analysis frame by frame. The durations of the movements measured during swallowing were: a) oral transit (OT): the tongue tip at incisors to the arrival of the bolus tail at the oral-pharyngeal transition; b) pharyngeal transit (PT): the bolus tail at the oral-pharyngeal transition to the bolus tail finish as it passed the upper esophageal sphincter (UES); c) pharyngeal clearance (PC): the bolus head at the oral-pharyngeal transition to the bolus tail as it finished passing the UES; d) UES opening (UESO): the time interval between the bolus head to enter the UES to the

’ RESULTS There was no effect of bolus volume on the duration of oral transit, pharyngeal clearance, hyoid movement and oralpharyngeal transit (p40.05). The pharyngeal transit duration (Figure 1) was shorter with the 10 mL bolus compared to the 5 mL bolus, for men and women (Table 2) and for taller and shorter individuals (Table 3). The differences between the mean pharyngeal transit duration for the 5 mL bolus and the mean pharyngeal transit duration for the 10 mL bolus were 35ms for men, 34ms for women, 39ms for taller subjects and 31ms for shorter subjects. The UES opening duration (Figure 2) was longer with the 10 mL bolus than with the 5 mL bolus, for men and women (Table 2) and for taller and shorter individuals (Table 3). The differences between the mean UES opening durations for the 5mL bolus and the 10mL bolus were 36ms for men, 27ms for women, 34ms for taller subjects and 29ms for shorter individuals.

’ DISCUSSION Men have a greater oral capacity than women to accommodate a volume of liquid (19), which may be attributed to the height differences between them. Measuring the volume of the oropharyngeal cavity, the volume of the laryngeal and hypopharyngeal cavities, the length and width of the pharynx and the volume of the pyriform sinus, found higher values for men than for women (13). This investigation demonstrated that the pharyngeal transit duration, measured from the time the bolus tail crossed the oral-pharyngeal transition to the time the bolus tail finished crossing the UES, was faster for a 10 mL liquid bolus than for a 5 mL liquid bolus. Although there are anatomic differences related to corporal height, and men and women may have some differences in swallowing dynamics, the results regarding the increase in bolus volume were similar. The faster pharyngeal bolus transit might be associated with the increase in upstream intrabolus pressure with the

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Figure 1 - Pharyngeal transit duration, in milliseconds (ms), measured as the time between the bolus tail arriving at the oral-pharyngeal transition and the time the bolus finished passing the upper esophageal sphincter (UES). pp0.05, 5 mL vs. 10 mL.

Table 2 - Oral and pharyngeal swallowing event durations (in milliseconds) in men (n=20) and women (n=20) after swallowing 5 and 10 mL liquid boluses. MEN 5 mL

OT PT PC UESO HM OPT

WOMEN 10 mL

5 mL

10 mL

Mean

95% CI

Mean

95% CI

Mean

95% CI

Mean

95% CI

718 403 636 355 979 1116

633-803 344-461 552-718 323-386 877-1082 1016-1213

672 368 621 391 894 1042

586-759 314-422 556-687 359-423 793-994 945-1139

0.17 0.05* 0.76 0.01* 0.44 0.26

954 272 448 290 811 1223

809-1100 234-309 425-472 274-306 706-916 1073-1373

900 238 465 317 862 1152

785-1015 219-257 433-496 299-335 777-946 1015-1288

0.52 0.04* 0.37 0.01* 0.27 0.27

OT – oral transit; PT – pharyngeal transit; PC – pharyngeal clearance; UESO – upper esophageal sphincter opening; HM – hyoid movement; OPT – oral-pharyngeal transit. * pp0.05, 5 mL vs. 10 mL.

Table 3 - Oral and pharyngeal swallowing event durations (in milliseconds) in taller (n=20) and shorter (n=20) subjects after swallowing 5 and 10 mL liquid boluses. TALLER 5 mL

OT PT PC UESO HM OPT

SHORTER 10 mL

5 mL

10 mL

Mean

95% CI

Mean

95% CI

Mean

95% CI

Mean

95% CI

914 368 562 329 867 1260

758-1070 304-432 487-637 303-354 738-996 1103-1417

857 329 564 363 902 1169

730-984 275-385 502-626 332-393 820-985 1038-1301

0.38 0.05* 0.52 0.01* 0.87 0.17

758 307 522 316 923 1079

686-830 273-340 467-577 289-342 847-1000 945-1164

716 276 522 345 853 1024

641-789 249-304 475-569 321-370 751-955 921-1127

0.26 0.04* 0.58 0.01* 0.08 0.40

duration might be the UES alteration with the increase in the bolus volume, with a decrease in the minimal UES pressure with the increase in the bolus volume (23), which could facilitate bolus propulsion and transit through the pharynx.

increase in the bolus volume (20-22), or a likely increase in the base of the tongue pressure. However, a previous investigation did not find alterations in the maximal tongue base pressure with the variation of bolus volumes (23). The explanation for the alteration of the bolus transit

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Figure 2 - Upper esophageal sphincter (UES) opening duration, in milliseconds (ms), measured as the time between the bolus head entering the UES and the bolus tail passing the UES. p=0.01, 5 mL vs. 10 mL.

volume of the thick liquid and of honey-like consistencies (4). Our results showed that the pharyngeal transit has a shorter duration with the 10 mL bolus than with the 5 mL bolus of liquid. The different conclusions of these investigations suggested that the bolus consistency and viscosity may influence the response of the pharyngeal function to the bolus volume. For the transit through the UES, the conclusions of the investigation, which compared 5 mL and 10 mL of thick liquid and honey-thick boluses, were similar in this study (4). Men and women, taller and shorter individuals, had similar body mass index, indicating that the observed differences should not be a consequence of the weight. This study has several limitations. A larger number of volunteers would be beneficial to clarify the differences that did not reach statistical significance. The inclusion of a bolus with paste consistency might exibit the influence of height and sex on the swallowing adaptation to the bolus volumes of different consistencies. The observation of swallowing modification with bolus volume should be considered by caregivers of patients with dysphagia. Modifying the bolus volume during eating may not be possible for some of them, because of the swallowing function limitations caused by the disease. The BMI values of the groups were very similar, and the influence of the weight in the results was not expected to occur. In conclusion, the results suggest that the increase in bolus volume from 5 mL to 10 mL causes an increase in the bolus transit duration through the UES and a decrease in the pharyngeal bolus transit duration, without differences caused by sex and corporal height.

Compared with a smaller volume, a greater bolus volume causes a longer time for the bolus to cross the UES (1,3,4,24), even with increased sphincter diameter and area when swallowing a larger volume (1,20,21). The liquid bolus length in the pharynx increases from 49.3(SE, 1.7)mm with the 5 mL bolus to 62.9(SE, 2.5)mm with the 10 mL bolus (1), and the maximum area of the UES opening, calculated as having an ellipse shape, increases from 181(SD, 59)mm2 with the 5 mL bolus to 195(SD, 45) mm2 with the 10 mL bolus (21). A 10 mL bolus arrived at the UES earlier than a 5 mL bolus and caused an early UES opening, without significantly changing the timing of the end of the bolus transit through the sphincter (21,24). The increase in bolus length and early bolus arrival at the UES can justify the increase in the duration until the bolus crossed the UES even with a faster bolus transit through the pharynx. Thus, a longer bolus length takes a longer time to cross the UES. A previous investigation did not describe differences in pharyngeal transit duration, pharyngeal peristaltic wave amplitude and duration, and upstream intrabolus pressure in the pharynx between the swallowed liquid bolus volumes of 5 mL and 10 mL (1). In different studies, the authors used different barium concentrations. Even small differences in barium concentrations may cause modifications in the timing of swallowing events (25,26), which might explain the different results observed in the different investigations. The new consistency classification proposed by the International Dysphagia Diet Standardisation Initiative (IDDSI) has clear terminology and definitions of consistencies of liquids and solid foods, which are important for treating patients with dysphagia and in padronization of bolus consistency used in the investigation of swallowing dynamics. The proposed method for evaluating liquid consistency is ease and cost-effectiveness, using a 10 mL syringe (16,27). A longer duration of pharyngeal transit was demonstrated only with an increased bolus volume (17.3 mL) compared to that with a smaller bolus volume (3.5mL) (3). No difference was observed between the 5 mL volume and the 10 mL

â&#x20AC;&#x2122; AUTHOR CONTRIBUTIONS Regueiro MR, Nascimento WV, Parreira LC and Dantas RO participated in the design of the study; collection, analysis and interpretation of the data; report writing and in making the decision to submit the study for publication.

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â&#x20AC;&#x2122; REFERENCES

15. Huang J, Shen H, Takahashi M, Fukunaga T, Toga H, Takahashi K, et al. Pharyngeal cross-sectional area and pharyngeal compliance in normal males and females. Respiration. 1998;65(6):458-68, http://dx.doi.org/ 10.1159/000029314. 16. Cichero JA, Lam P, Steele CM, Hanson B, Chen J, Dantas RO, et al. Development of International Terminology and Definitions for TextureModified Foods and Thickened Fluids Used in Dysphagia Management: The IDDSI Framework. Dysphagia. 2017;32(2):293-314, http://dx.doi. org/10.1007/s00455-016-9758-y. 17. Santos CM, Cassiani RA, Dantas RO. Videofluoroscopic evaluation of swallows in anorexia nervosa. Arq Gastroenterol. 2016;53(3):136-40, http://dx.doi.org/10.1590/S0004-28032016000300003. 18. Schall R. Estimation in generalized linear models with random effects. Biometrika. 1991;78(4):719-27, http://dx.doi.org/10.1093/biomet/78.4.719. 19. Nascimento WV, Cassiani RA, Dantas RO. Gender effect on oral volume capacity. Dysphagia 2012;27(3):384-9, http://dx.doi.org/10.1007/s00455011-9379-4. 20. Cock C, Jones CA, Hammer MJ, Omari TI, McCulloch TM. Modulation of upper esophageal sphincter (UES) relaxation and opening during volume swallowing. Dysphagia. 2017;32(2):216-24, http://dx.doi.org/10.1007/ s00455-016-9744-4. 21. Cook IJ, Dodds WJ, Dantas RO, Massey B, Kern MK, Lang IM, et al. Opening mechanisms of the human upper esophageal sphincter. Am J Physiol. 1989;257(5 Pt 1):G748-59. 22. Ghosh SK, Pandolfino JE, Zhang Q, Jarosz A, Kahrilas PJ. Deglutitive upper esophageal sphincter relaxation: a study of 75 volunteers subjects using solid state high-resolution manometry. Am J Physiol Gastrointest Liver Physiol. 2006;291(3):G525-31, http://dx.doi.org/10.1152/ajpgi. 00081.2006. 23. Ryu JS, Park D, Oh Y, Lee ST, Kang JY. The effects of bolus volume and texture on pharyngeal pressure events using high-resolution manometry and its comparison with videofluoroscopic swallowing study. J Neurogastroenterol Motil. 2016;22(2):231-9, http://dx.doi.org/10.5056/ jnm15095. 24. Cook IJ, Dodds WJ, Dantas RO, Kern MK, Massey BT, Shaker R, et al. Timing of videofluoroscopic, manometric events, and bolus transit during the oral and pharyngeal phases of swallowing. Dysphagia. 1989;4(1):8-15, http://dx.doi.org/10.1007/BF02407397. 25. Dantas RO, Dodds WJ, Massey BT, Kern MK. The effect of high- vs lowdensity barium preparations on the quantitative features of swallowing. AJR Am J Roentgenol. 1989;153(6):1191-5, http://dx.doi.org/10.2214/ ajr.153.6.1191. 26. Stokely SL, Molfenter SM, Steele CM. Effects of barium concentration on oropharyngeal swallow timing measures. Dysphagia. 2014;29(1):78-82, http://dx.doi.org/10.1007/s00455-013-9485-6. 27. Hanson B. A review of diet standardization and bolus rheology in the management of dysphagia. Curr Opin Otolaryngol Head Neck Surg. 2016;24(3):183-90, http://dx.doi.org/10.1097/MOO.0000000000000251.

1. Dantas RO, Kern MK, Massey BT, Dodds WJ, Kahrilas PJ, Brasseur JG, et al. Effect of swallowed bolus variables on oral and pharyngeal phase of swallowing. Am J Physiol. 1990;258(5 Pt 1):G675-81. 2. Mendell DA, Logemann JA. Temporal sequence of swallow events during oropharyngeal swallows. J Speech Lang Hear Res. 2007;50(5):1256-71, http://dx.doi.org/10.1044/1092-4388(2007/088). 3. Molfender SM, Steele CM. Variation in temporal measures of swallowing: sex and volume effects. Dysphagia. 2013;28(2):226-33, http://dx.doi.org/ 10.1007/s00455-012-9437-6. 4. Nascimento WV, Cassiani RA, Santos CM, Dantas RO. Effect of bolus volume and consistency on swallowing events duration in healthy subjects. J Neurogastroenterol Motil. 2015;21(1):78-82, http://dx.doi.org/ 10.5056/jnm14055. 5. Pearlman AL, Schultz PG, VanDaele DJ. Effects of age, gender, bolus volume, and bolus viscosity on oropharyngeal pressure during swallowing. J Appl Physiol. 1993;75(1):33-7. 6. Dantas RO, de Aguiar Cassiani R, dos Santos CM, Gonzaga GC, Alves LM, Mazin SC. Effect of gender on swallow event duration assessed by videofluoroscopy. Dysphagia. 2009;24(3):280-4, http://dx.doi.org/10.1007/ s00455-008-9202-z. 7. Alves LM, Cassiani RA, Santos CM, Dantas RO. Gender effect on the clinical measurement of swallowing. Arq Gastroenterol. 2007;44(3):227-9, http://dx.doi.org/10.1590/S0004-28032007000300009. 8. Hughes TA, Liu P, Griffiths H, Wiles CM. The repeatability and variability of electrical impedance tomography indices of pharyngeal transit time in normal adults. Physiol Meas. 1995;16(3 Suppl A):A79-86. 9. Hughes TA, Wiles CM. Clinical measurement of swallowing in health and in neurogenic dysphagia. QJM. 1996;89(2):109-16, http://dx.doi.org/ 10.1093/qjmed/89.2.109. 10. Robbins J, Hamilton JW, Lof GL, Kempster GB. Oropharyngeal swallowing in normal adults of different ages. Gastroenterology. 1992;103(3):823-9, http://dx.doi.org/10.1016/0016-5085(92)90013-O. 11. Kim Y, McCullough GH, Asp CW. Temporal measurements of pharyngeal swallowing in normal populations. Dysphagia. 2005;20(4):290-6, http:// dx.doi.org/10.1007/s00455-005-0029-6. 12. Molfenter SM, Steele CM. Use of an anatomical scalar to control for sexbased size differences in measures of hyoid excursion during swallowing. J Speech Lang Hear Res. 2014;57(3):768-78, http://dx.doi.org/10.1044/ 2014_JSLHR-S-13-0152. 13. Inamoto Y, Saitoh E, Okada S, Kagaya H, Shibata S, Baba M, et al. Anatomy of the larynx and pharynx: effects of age, gender and height revealed by multidetector computed tomography. J Oral Rehab. 2015; 42(9):670-7, http://dx.doi.org/10.1111/joor.12298. 14. Daniel MM, Lorenzi MC, da Costa Leite C, Lorenzi-Filho G. Pharyngeal dimensions in healthy men and women. Clinics. 2007;62(1):5-10, http:// dx.doi.org/10.1590/S1807-59322007000100002.

697

CLINICAL SCIENCE

A novel body mass index reference range - an observational study Sirlei Siani Morais, * Mirena Ide, Andrea Moreno Morgan, Fernanda Garanhani Surita Departamento de Ginecologia e Obstetricia, Faculdade de Ciencias Medicas, Universidade de Campinas (UNICAMP), Campinas, SP, BR.

OBJECTIVE: To generate a new body mass index curve of reference values and ranges for body mass index and weight gain during pregnancy and to compare the new curve and weight gain ranges with the currently used references. METHODS: A prospective observational study was conducted with a total of 5,656 weight and body mass index measurements in 641 women with single pregnancy who attended their first prenatal visit before 12 weeks. All the women were over 18 years old and had no medical conditions that would influence body mass index. Data were collected using prenatal charts and medical records during hospitalization for childbirth. A linear regression method was used for standard curve smoothing in the general population and for specific curves according to the baseline body mass index classification. Curves were obtained for the 5th, 10th, 50th, 85th, 90th and 95th percentiles. Concordance between the classification of women using the newly generated and currently used curves was evaluated by percentages and kappa coefficients. The weight gain was compared with the reference values of the Institute of Medicine using Student’s T test. The data were analyzed using SAS software version 9.2, and the significance level was set at 5%. RESULTS: A general reference curve of percentiles of body mass index by gestational age was established. Additionally, four specific curves were generated according to the four baseline body mass index categories. The new general curve offered percentile limits for women according to their initial body mass index and according to the Centers for Disease Control and Prevention limits, showing poor agreement with the currently used curve (48.3%). Women who were overweight or obese when starting prenatal care had higher weight gain than the Institute of Medicine recommendation. CONCLUSIONS: The new proposed curve for body mass index during pregnancy showed weak agreement with the currently used curve. The new curve provided more information regarding body mass index increase using percentiles for general and specific groups of body mass index. Overweight pregnant women showed an upward body mass index trend throughout pregnancy that increased more dramatically than those of other groups of pregnant women, and they also presented a major mean difference between weight gain and the Institute of Medicine recommendation. KEYWORDS: Gestational Weight Gain; Body Mass Index; Prenatal Care. Morais SS, Ide M, Morgan AM, Surita FG. A novel body mass index reference range - an observational study. Clinics. 2017;72(11):698-707 Received for publication on January 24, 2017; First review completed on May 22, 2017; Accepted for publication on September 4, 2017 *Corresponding author. E-mail: surita@unicamp.br

’ INTRODUCTION

Several recommendations for gestational WG have been adopted in different populations based on different parameters, such as weight or body mass index (BMI) before pregnancy and distribution curves of increased BMI or WG ranges during pregnancy. However, because of intense sociocultural and behavioral changes in the last few decades, it is necessary to update the scientific knowledge and the normal range for these recommendations and, therefore, to establish parameters for health professionals to guide pregnant women (7,8). These recommendations differ among countries. In Sweden, Germany, Switzerland, Austria and Turkey, the recommendations are based on caloric intake. The Institute of Medicine (IOM) in the United States suggests a weekly WG range by trimester based on pre-pregnancy BMI. WG recommendations according to pre-pregnancy BMI are followed by other countries (Italy, Vietnam, Western European countries,

Nutritional disorders have become a worldwide problem due to the high prevalence of obesity during different stages of life. For women of reproductive age, two extreme conditions should be considered: on the one hand, obesity and excessive gestational weight gain (WG), and on the other hand, women with insufficient WG who compulsively avoid increased weight during pregnancy (1-6).

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849 clinical records were assessed, among which 753 met the inclusion criteria and 641 contained complete information as required by the study protocol. The records showed that the prenatal charts contained between 6-16 weight measurements, resulting in 5,656 weight measurements at different gestational ages. The inclusion criteria were verified with a checklist, and the available data were transcribed into a specific form and stored in an Excel file. Double entry of the data was performed, and then the data were validated in Excel. After a detailed consistency checking procedure, inconsistencies in the database were reassessed using the data collection form, clinical records and prenatal charts as the main sources of data.

Australia and Pacific Islands). The total WG forecast is also used in clinical practice. Increased caloric intake until the end of pregnancy is recommended in Japan. Singapore uses WG according to height, and the Philippines, India and Sudan use a recommendation of simply gaining weight and following a good diet (7). In Brazil, the Ministry of Health suggests the use of Atalah’s curve (9), which incorporates the intersection of BMI and gestational age and has the advantage of not requiring a standard reference. BMI use simplifies nutritional assessments during pregnancy, is easy to calculate and demonstrates a good association with the degree of adiposity and the risk of non-communicable chronic diseases (9,10). However, the use of Atalah’s curve in Brazil should consider the pre-pregnancy BMI classification, which differs from the current WHO classification. The curve was developed a few decades ago; it does not provide clinical parameters to evaluate upper and lower limits for obese and underweight women and was developed from a crosssectional study of Chilean women (7,9,11). The purpose of the present study was to assess BMI during pregnancy using a longitudinal study of Brazilian pregnant women to generate a reference curve according to prepregnancy BMI and, therefore, to provide new parameters that could be used to monitor the weight of women during pregnancy.

Data analyses For maternal and perinatal data assessments, absolute and relative frequencies were used for the sample of 641 women. They were classified according to the first BMI evaluation during prenatal care, as defined by the WHO criteria using the weight/height2 formula, into four categories: low weight (o18.5 kg/m2), adequate weight (18.5 to o25.0 kg/m2), overweight (25.0 to o30 kg/m2) and obese (X30 kg/m2) (16). The weight measurement was first assigned using the dependent data; however, because the measurements were collected from prenatal charts, there were substantial missing data at many gestational ages. Therefore, the values were studied in independent form: each gestational age and weight was considered one measurement of the sampling unit. All the measurements and a stratification of the sample according the classes of initial BMI (WHO criteria) were evaluated to determine the equation to describe the evaluation of the change in weight and BMI according to gestational age during pregnancy using a simple linear regression, and they were found to be normally distributed. However, the coefficient determination (R2) estimated for the curve by linear regression was low, and the curves were considered to have low predictive value. The 5th, 10th, 50th, 85th, 90th and 95th percentiles for gestational age for the entire sample and for initial BMI stratification were described. A simple linear regression was also used for these new values to smooth the curve, and the equation was used to estimate the reference values for each percentile. The smoothed equation of the percentiles that was obtained from the entire sample curve was then used to classify each of the 641 women during early pregnancy (first measure), in the middle of pregnancy (between 19-23 weeks) and at the last prenatal evaluation (between 35 to 41 weeks). We classified the women according to four categories using the same percentiles as the Centers for Disease Control and Prevention (CDC) (17): low weight (those with BMI oP5), adequate weight (BMI XP5 and BMI oP85), overweight (BMI XP85 and BMI oP95) and obese (BMI 4P95). The mean WG during pregnancy was then compared with the IOM recommendation, and the difference was calculated and compared using Student’s T test. The women were also classified by their BMI using two different instruments, the newly proposed curve and Atalah’s curve, during the three different periods of pregnancy: early, middle and late. The classification by Atalah’s curve was then compared with the classification by the new curve to assess prominent modifications. McNemar’s test and the weighted kappa coefficient, with its respective confidence intervals, were used. The significance level was 5%, and the data were

’ MATERIALS AND METHODS The research protocol for this study was approved by the Institutional Review Board of the School of Medical Sciences of the University of Campinas and took into account all the requirements established by the Brazilian National Health Council. A prospective observational study was conducted at the State Hospital of Sumaré (SHS), a university teaching hospital that is a reference for low-risk pregnant women and is affiliated with the University of Campinas. This hospital is located in the metropolitan region of Campinas, state of São Paulo, Brazil. The state of São Paulo has the highest population density in the country, with an estimated population of 41 million, and the metropolitan region of Campinas has the third largest population of women of reproductive age (12,13). From March to October 2015, data were collected from prenatal charts and the medical records of women who gave birth at the SHS. Women who had some conditions that could influence BMI during pregnancy, such as diabetes, drug use, HIV infection, multiple pregnancy or cancer, as well as women without prenatal care, with onset of prenatal care after 16 weeks of gestational age or women under 18 years of age, were excluded. The sample size was calculated to evaluate the BMI variation with a representative number of weight measurements in all gestational ages. We used a reported BMI mean value of 24.2±4.5 kg/m2 in pregnancy. Considering a significance level of 5% and variation of 2%, the sample size was estimated to be 333 women (to evaluate the BMI variation) (14). To evaluate the measurement, it was estimated that most women had approximately six prenatal visits during pregnancy. According to data from SHS in 2009, for the 2,340 deliveries that occurred there, an average number of 14,040 measurements would be available (15). Considering a significance level of 5% and a sampling error of 2%, the sample size was calculated as 2,050 measurements. A total of

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Women classified at the first prenatal visit as low weight or adequate weight did not present any significant difference between their gestational WG during pregnancy and the IOM recommendation. Women classified as overweight at the first prenatal visit had a mean WG of 4.1 kg above that recommended by the IOM, and those classified as obese had a mean WG of 2.2 kg above the IOM recommendation. Using the equations presented in Table 3, we can estimate the percentiles of BMI for any gestational age. However, we estimated the 5th, 10th, 50th, 85th, 90th and 95th percentiles for each gestational age to evaluate the BMI of the pregnant women using the equation for each gestational age between 8 and 40 weeks. The reference values for these percentiles are available in Table 4 or were alternatively generated using an Excel file calculator provided by the authors at https://www.dropbox.com/s/mbsfldtv257n6zn/ calculadora%20-%20link.xlsx?dl=0. According to the obtained reference value data, a general curve was created. Four other specific curves were generated according pregestational BMI to monitor WG and consequent changes in BMI during pregnancy (figures 1 to 5). The ranges of BMI values between percentiles 50 and 90 or between 50 and 95 were larger than the ranges of BMI between percentiles 5 and 50 or 10 and 50, showing a trend toward a higher concentration of cases in the upper part of the curve. The four smoothed curves generated according to the first prenatal BMI were similar to the general curve at some points. However, there were different slopes by initial BMI. The slope of the line generated for the 50th percentile was higher among women with a normal weight (slope=0.167), followed by overweight (slope=0.159), low-weight (slope=0.156) and obese women (slope=0.115), demonstrating that the majority of

analyzed using SAS version 9.4 software Copyright (c) 20022012 by SAS Institute Inc., Cary, NC, USA. All STROBE statement items for a prospective study were followed and checked in this manuscript (18). Financial support for the current study was obtained from the SĂŁo Paulo Research Foundation (FAPESP), grant number 2014/01770-7. The content of this article is solely the responsibility of the authors and does not necessarily represent the official views of FAPESP, which did not influence the content of the manuscript.

â&#x20AC;&#x2122; RESULTS A total of 5,656 weight and BMI measurements from 641 women with single pregnancy and first prenatal visit before 16 weeks were obtained. Most of the women had white skin color (62%), were 20-34 years old (76.5%), and were primiparas (74.5%), and maternal anemia was present in 15.3% of the sample. Prenatal care was started between 6 and 12 weeks in 72.9% and between 12 and 16 weeks in 23.6% of the women. The first BMI measurement during prenatal care classified 47.1% of the women as adequate weight, 30.9% as overweight, and 16.7% as obese, resulting in a total of 47.5% of women with excessive weight. Vaginal delivery occurred in 59.8% of all pregnancies. Neonatal results showed that most newborns weighed 2,500-4,000 g (86.7%). Macrosomia occurred in 5.1% of newborns. Complete data with some characteristics of the sample are presented in Table 1. The mean increase in BMI during gestation was 2.7 to 4.6 units, representing a percentage mean increase in BMI between 8.2 and 26.1%. Of the women classified as obese at the first prenatal visit, 33.7% had excessive gestational WG according to the IOM recommendation (Table 2).

Table 1 - Sociodemographic, nutritional, delivery and neonatal characteristics of the sample. Maternal characteristics Ethnicity (n = 619) White Non-White Age (n = 639) Mean (SD) p29 30-39 X40 Parity (n = 638) Primiparous Multiparous Maternal anemia (n = 590) Yes No Missing Marital Status (n = 619) With partner Without partner Gestational age at first prenatal visit (weeks) up to 5 6 to 12 12 to 16 Mean (SD) Initial BMI (kg/m2) Low weight (o18.5) Adequate weight (18.5-24.99) Overweight (25-29.99) Obese (X30) Mean (SD)

(n = 641)

396 243

62.0 38.0

26 457 171 11

(5.7) 71.5 26.8 1.7

493 165

74.9 25.1

93 468 49

15.3 76.7 8.0

465 174

72.8 27.2

23 482 156 9.9

3.5 72.9 23.6 (2.8)

36 311 204 110 25.3

5.5 47.1 30.9 16.9 (5.2)

Delivery and neonatal characteristics Mode of delivery (n = 634) C-section Vaginal Episiotomy (n = 391) Yes No Birthweight (n = 628) (g) o2500 2500 -3999 X4000 Adequacy of birthweight (n = 627) SGA AGA LGA APGAR 1st minute (n = 623) o7 X7 APGAR 5th minute (n = 623) o7 X7 Somatic neonatal age (n = 606) (weeks) o 37 X 37

SD(standart desviation),SGA (small for gestational age), AGA (adequate for gestational age), LGA (large for gestational age).

700

(n = 641)

263 391

40.2 59.8

208 181

53.5 46.5

53 562 33

8.2 86.7 5.1

36 539 72

5.6 83.3 11.1

62 581

9.6 90.4

2 641

0.3 99.7

51 575

8.2 91.9

A novel body mass index reference range Morais SS et al.

0.6324 0.6695 o 0.0001 o 0.0001

women (50%) who initially gained weight had an adequate weight or were overweight (Table 3). The curve of the women with low weight (figure 2) displayed a greater dispersion of BMI values during the final weeks than in the first week of prenatal care. The mean WG in these women with low weight was 12.1±4.7 kg, ranging between 0.80 kg and 24.9 kg. The average percentage of WG was 26.1±10.6%, ranging from 1.6% to 51.7% (data not shown). In women with an adequate initial BMI, the new curve (figure 3) showed that BMI increases occurred proportionally throughout gestation, as shown by the parallel percentile lines. The average WG of women with an adequate BMI was 11.5±5.0 kg. The mean percentage of WG was 20.3±9.0% (data not shown). For women classified as overweight, the slopes of the percentiles were mostly lower than those of the low-weight, adequate-weight and obese women (figure 4). The average WG (in kg) of overweight women was 10.1±6.4 kg, ranging from -10.6 kg to 28.2 kg. The average percentage of WG was 14.5%±9.5% (data not shown). Obese women displayed a curve with a broad slope (figure 5), but an increase in data dispersion (variation of percentiles around the smoothed line) and a greater range were observed compared with those in the other groups. The average WG percentage was 7.2%±5.8%, ranging from -9.4% to 28.8% (data not shown). The agreement between the new curve and Atalah’s curve was approximately 50%. Among the 51.7% of women with discordant data in early pregnancy, 39.8% (27.7% overweight/adequate + 12.1% obese/overweight) represented an underestimation of Atalah’s curve compared with the new curve for women classified as underweight or overweight by Atalah’s curve. In the middle of pregnancy, the most discordant findings (25.8%) were for women classified as overweight by Atalah’s curve and adequate by the new curve. At the end of the prenatal period, this percentage was 26.7%. There was complete disagreement regarding women classified as overweight by Atalah’s curve. In general, Atalah’s curve provided classifications with good agreement for adequate women but with disagreement for low-weight and overweight women. The agreement could be considered weak for all the evaluated data based on the values obtained by the weighted kappa (Table 5).

11.8 15.7 38.2 33.7 12.1 11.5 10.1 7.2 10.0 9.0 9.5 6.5

26.1 20.3 14.5 8.2 4.6 4.4 3.9 2.7 Low weight Adequate weight Overweight Obesity

36 311 204 110

5.5 47.1 46.1 16.6

1.8 1.9 2.5 2.2

mean

* The initial weight was considered the weight at the first prenatal care visit (max: 12 weeks). ** Percentage of women with weight gain greater than the IOM recommendation, according to the original BMI classification. WHO = World Health Organization. BMI = body mass index. SD = standard deviation. IOM = Institute of Medicine. p-value = T test of mean weight gain during gestation and respective mean of IOM recommendation.

4.7 5.0 6.4 5.8

12.5 11.5 7.0 5.0

18.0 16.0 11.5 9.0

IOM recommendation mean %

BMI increase in gestation*

SD mean % N

WHO BMI classification at first prenatal visit

Table 2 - Gestational weight gain according to initial BMI and comparison with the IOM recommendations.

Weight gain in gestation*

% of extra IOM**

p-value

CLINICS 2017;72(11):698-707

’ DISCUSSION The results of this study add reference values for BMI throughout gestation, both with regard to the lower limit through the 5th and 10th percentile curves and to the upper limits through the 90th and 95th percentile. The smoothed curves provide health teams with clinical tools to evaluate the BMI of pregnant women at each gestational age throughout pregnancy. A pregnant woman’s BMI values can be evaluated using the curve of the general population and, more specifically, through the different curves established according to BMI in early pregnancy using the WHO criteria (four categories). The standard increase in BMI during pregnancy was specific to each classification of early BMI in prenatal care, as observed in other studies (11,19,20). The new curves showed weak agreement with the current curve (Atalah’s). However, an overestimation of the values at the beginning of prenatal care was observed, which is consistent with previous comparisons with Atalah’s curve

701

P5 P 10 P 50 P 85 P 90 P 95

Adequate Gain

Adequate gain

Adequate Gain

702

Adequate Gain

= = = = = =

30.0 30.3 32.8 37.9 39.0 40.6

23.8 24.0 24.9 26.6 27.0 27.0

17.2 17.4 20.2 22.1 22.3 22.6

15.1 14.8 16.2 16.1 16.5 16.1

16.8 17.4 22.9 28.8 31.3 34.3

Intercept

18.6 19.4 24.8 30.6 32.9 35.8 16.4 16.4 18.1 18.6 19.0 19.0 18.9 19.4 22.2 24.2 24.6 25.1 24.9 25.3 26.8 28.8 29.2 29.6 30.5 31.1 34.2 38.7 39.7 41.5

0.90 0.96 0.93 0.79 0.62 0.80

Low Weight (Initial BMI o18.5 kg/m2) 0.110 X GA 0.57 0.133 X GA 0.73 0.156 X GA 0.93 0.212 X GA 0.93 0.207 X GA 0.85 0.240 X GA 0.83 Adequate Weight (18.5 p Initial BMI o25 kg/m2) 0.145 X GA 0.93 0.163 X GA 0.96 0.167 X GA 0.93 0.178 X GA 0.94 0.190 X GA 0.95 0.207 X GA 0.95 Overweight (25 p Initial BMI o30 kg/m2) 0.090 X GA 0.85 0.106 X GA 0.92 0.159 X GA 0.94 0.183 X GA 0.93 0.186 X GA 0.94 0.214 X GA 0.93 Obese (Initial BMI X30 kg/m2) 0.040 X GA 0.35 0.070 X GA 0.55 0.115 X GA 0.72 0.070 X GA 0.14 0.061 X GA 0.09 0.079 X GA 0.12

+ + + + + + + + + + + + + + + + + + + + + + + +

30.8 31.7 35.1 39.3 40.2 42.2

25.6 26.1 28.1 30.3 30.7 31.3

20.1 20.7 23.5 25.7 26.1 26.7

17.3 17.5 19.3 20.3 20.6 20.9

19.7 20.8 26.0 31.8 34.0 36.7

31.1 32.3 36.0 39.9 40.7 42.8

26.3 27.0 29.4 31.7 32.2 33.0

21.3 22.0 24.9 27.1 27.6 28.4

18.2 18.5 20.6 22.0 22.3 22.8

20.9 22.1 27.2 33.0 35.1 37.7

BMI (kg/m2) of percentile example for GA (weeks)

0.147 0.168 0.155 0.151 0.134 0.122

R2 of model

+ + + + + +

Any Initial BMI X GA X GA X GA X GA X GA X GA

Slope X GA

GA = gestational age, BMI = body mass index. P = percentile, R2 = determination coefficient. Weight gain: underweight = any BMI value lower than P5; Obesity = any BMI value greater than P95.

Excessive Gain

P5 P 10 P 50 P 85 P 90 P 95

Percentile

Adequate Gain

Weight Gain

Table 3 - Equations for generation of smoothed curves of BMI percentiles during pregnancy for all women and according to initial BMI.

A novel body mass index reference range Morais SS et al. CLINICS 2017;72(11):698-707

703

9 7 5 8 6 7 5 7 14 6 4 6 7 11 7 8 4 10 6 6 7 6 11 10 8 10 12 13 13 15 28 25 11 5

o8 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

17.7 17.7 17.5 17.2 18.1 17.8 17.8 18.7 17.7 18.5 18.1 19.0 19.0 18.6 18.6 19.6 18.8 19.5 20.6 20.8 20.6 19.6 20.0 20.9 21.9 20.4 20.8 21.3 22.4 21.7 22.1 22.3 23.2 23.1

Mean

0.53 0.96 1.02 1.15 0.68 1.31 1.05 1.77 1.13 0.37 0.97 1.36 0.65 1.12 1.50 1.28 1.44 1.29 0.73 1.30 1.59 1.00 1.64 1.34 1.69 2.31 2.08 1.40 1.70 1.63 1.86 2.00 2.17 2.35

15.9 16.0 16.1 16.2 16.3 16.4 16.5 16.7 16.8 16.9 17.0 17.1 17.2 17.3 17.4 17.5 17.6 17.8 17.9 18.0 18.1 18.2 18.3 18.4 18.5 18.6 18.8 18.9 19.0 19.1 19.2 19.3 19.4 19.5

P5% 17.8 18.0 18.2 18.5 18.7 19.0 19.2 19.4 19.7 19.9 20.2 20.4 20.6 20.9 21.1 21.4 21.6 21.8 22.1 22.3 22.6 22.8 23.0 23.3 23.5 23.8 24.0 24.2 24.5 24.7 25.0 25.2 25.4 25.7

P95% 88 56 41 55 55 58 58 53 76 47 67 56 62 61 74 55 53 69 70 62 58 66 65 81 74 92 81 104 122 137 165 172 119 45

n 22.1 22.0 21.8 22.1 22.0 22.2 21.8 22.2 22.1 22.4 22.6 23.0 23.1 23.0 23.4 23.9 23.2 24.1 24.2 24.4 24.1 25.0 24.9 25.1 24.9 25.6 25.6 26.0 26.0 26.4 26.6 26.6 26.6 27.2

Mean 1.77 1.90 2.07 1.88 1.91 1.97 1.76 2.16 1.93 2.31 1.67 1.87 1.93 2.23 2.11 2.08 2.00 2.11 2.08 1.96 1.85 2.10 2.29 2.04 2.28 2.28 2.28 2.17 2.16 2.50 2.33 2.24 2.18 2.43

SD 18.3 18.4 18.5 18.7 18.8 19.0 19.1 19.3 19.4 19.6 19.7 19.9 20.0 20.1 20.3 20.4 20.6 20.7 20.9 21.0 21.2 21.3 21.5 21.6 21.7 21.9 22.0 22.2 22.3 22.5 22.6 22.8 22.9 23.1

P5% 24.0 24.2 24.4 24.6 24.8 25.0 25.2 25.4 25.6 25.9 26.1 26.3 26.5 26.7 26.9 27.1 27.3 27.5 27.7 27.9 28.1 28.3 28.5 28.7 29.0 29.2 29.4 29.6 29.8 30.0 30.2 30.4 30.6 30.8

P95%

Adequate Weight (18.5p Initial BMI o25 kg/m2)

P5% - 5th Percentile - Inferior Limit; P95% - 95th Percentile - Superior Limit.

Low Weight (Initial BMI o18.5 kg/m2)

55 42 27 35 28 46 45 50 49 37 37 43 54 34 38 48 43 49 35 48 52 45 32 53 52 62 68 62 79 107 125 99 86 38

n 26.3 27.1 27.4 26.7 27.1 26.9 27.3 27.0 26.9 27.2 27.4 28.0 27.6 27.9 28.7 28.4 28.6 28.8 28.7 29.4 29.2 29.3 29.1 29.8 30.1 30.0 30.1 30.2 30.7 30.7 31.2 31.1 31.2 31.0

Mean 2.18 1.37 1.66 1.51 1.68 1.58 1.59 1.58 1.82 1.73 1.46 1.76 1.78 1.61 2.01 1.81 1.86 1.45 2.00 1.81 2.19 3.21 1.68 2.27 2.03 1.98 2.18 2.24 2.30 2.22 3.01 2.48 2.75 2.52

SD 24.4 24.5 24.6 24.7 24.8 24.9 25.0 25.1 25.2 25.3 25.4 25.5 25.6 25.7 25.8 25.8 25.9 26.0 26.1 26.2 26.3 26.4 26.5 26.6 26.7 26.8 26.9 27.0 27.1 27.2 27.3 27.4 27.5 27.6

P5% 28.5 28.7 28.9 29.1 29.3 29.6 29.8 30.0 30.2 30.4 30.6 30.8 31.1 31.3 31.5 31.7 31.9 32.1 32.3 32.5 32.8 33.0 33.2 33.4 33.6 33.8 34.0 34.3 34.5 34.7 34.9 35.1 35.3 35.5

P95%

Overweight (25p Initial BMI o30 kg/m2)

Table 4 - Reference values of BMI according to initial BMI classification: low-weight and adequate-weight pregnant women.

26 11 21 23 18 19 18 26 33 22 21 14 21 27 25 18 22 25 21 29 18 19 29 31 31 33 32 32 38 54 57 53 49 17

n 35.2 35.4 34.1 35.0 34.3 35.0 35.3 34.1 34.8 34.1 35.2 35.5 35.8 34.7 34.4 37.9 36.7 35.6 34.8 35.5 36.3 36.2 36.0 36.1 36.7 36.1 36.9 36.4 37.4 37.5 37.5 37.4 36.1 36.6

Mean 4.14 3.86 3.37 3.42 3.38 4.98 2.83 3.49 3.51 2.94 4.63 2.61 3.97 2.90 2.55 4.48 4.25 2.45 4.19 3.25 3.08 3.59 3.32 3.44 3.47 2.57 3.42 2.96 3.51 3.69 3.45 3.21 3.30 3.79

30.3 30.4 30.4 30.4 30.5 30.5 30.6 30.6 30.7 30.7 30.8 30.8 30.8 30.9 30.9 31.0 31.0 31.1 31.1 31.2 31.2 31.2 31.3 31.3 31.4 31.4 31.5 31.5 31.6 31.6 31.6 31.7 31.7 31.8

P5%

41.2 41.3 41.4 41.4 41.5 41.6 41.7 41.8 41.8 41.9 42.0 42.1 42.1 42.2 42.3 42.4 42.5 42.5 42.6 42.7 42.8 42.9 42.9 43.0 43.1 43.2 43.2 43.3 43.4 43.5 43.6 43.6 43.7 43.8

P95%

Obese (Initial BMI X30 kg/m2)

CLINICS 2017;72(11):698-707 A novel body mass index reference range Morais SS et al.

A novel body mass index reference range Morais SS et al.

CLINICS 2017;72(11):698-707

Figure 1 - Percentiles and smoothed curves of BMI by gestational age – total sample (any initial BMI).

Figure 2 - Percentiles and smoothed curves of BMI by gestational age for women classified as low weight at the first prenatal visit.

Figure 3 - Percentiles and smoothed curves of BMI by gestational age for women classified as adequate weight at the first prenatal visit.

then, there have been changes in behavior and gestational weight recommendations, as well as cultural and racial influences that might impact the results in different countries (16,23). The results were based on a specific population with certain socio-demographic and cultural characteristics, which could be considered a standard low-risk population of pregnant women. These women had the expected nutritional

and is likely due to ethnic and cultural differences between Atalah’s original population and the Brazilian sample (21,22). Although it is the standard and recommended by the Ministry of Health in Brazil, Atalah’s curve has limitations, such as a lack of upper limits for pregnant women classified as obese and lower limits for pregnant women classified as underweight (9,12). Atalah’s curve was developed in the 1990s; since

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Figure 4 - Percentiles and smoothed curves of BMI by gestational age for women classified as overweight at the first prenatal visit.

Figure 5 - Percentiles and smoothed curves of BMI by gestational age for women classified as obese at the first prenatal visit.

Table 5 - Concordance between BMI classifications during pregnancy by Atalah’s curve and the proposed new curve (for all women). Curves Atalah’s / Proposed Curve Concordant Low Weight Adequate Weight Overweight Obesity TOTAL of concordant Discordant Adequate / Low Weight Overweight / Adequate Overweight / Obesity Obesity / Overweight Adequate / Overweight Low Weight / Adequate TOTAL of discordant McNemar’s p-value kappa (CI)

Beginning of prenatal period n

Middle (19 - 23 weeks) %

Last prenatal visit %

26 252 0 22 300

4.2 40.6 0.0 3.5 48.3

37 267 0 29 333

5.6 40.3 0.0 4.4 50.2

30 233 0 57 320

4.5 35.2 0.0 8.6 48.3

3 172 71 75 0 0 321

0.5 27.7 11.4 12.1 0.0 0.0 51.7

9 171 67 83 0 0 330

1.4 25.8 10.1 12.5 0.0 0.0 49.8

50 177 45 70 0 0 342

7.6 26.7 6.8 10.6 0.0 0.0 51.7

o0.0001 0.36 (0.32-0.41)

o0.0001 0.39 (0.35-0.44)

o0.0001 0.37 (0.33-0.42)

CI = confidence interval.

these results would need to be validated with data from larger samples and from different populations. This method could minimize the influence of factors such as ethnicity,

status for a middle-income setting, a low rate of anemia and a proportion of overweight women similar to those previously described. To be applied at a national or higher level,

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Guidelines are extremely important to support health care professionals in treating pregnant women and newborns, but we must consider the difficulties inherent in personal care, such as personal motivations and psychological support, that mathematical curves do not reflect. Thus, using curves is a simple method to professionally assess gestational WG in an individualized way. In Brazil specifically, this practice has been part of the prenatal routine for many years; our suggestion is that the currently used curve be replaced by the new proposed curve because it was created for Brazilian women. Therefore, a BMI classification of a pregnant woman can be provided according to gestational age, and assessments of the changes in her BMI can be compared to standard percentiles. These new curves seem to be useful and provide another tool for health professionals to monitor maternal health. In addition, depending on the provider’s decision, they can follow only women who are in the upper or lower limits for WG (11,23,30). The new curves developed in this work showed weak agreement with Atalah’s curve and provided additional information regarding BMI growth percentiles for general and specific body compositions. Women classified as overweight at the first prenatal visit had higher than recommended WG and an upward trend of BMI throughout pregnancy that was sharper than for the majority of pregnant women, indicating the need for a special focus on overweight women beginning in early prenatal care.

nutritional status or educational level. Hopefully, the present study population could be considered a representative sample of Brazilian pregnant women providing results that should be considered for public health purposes (12,24). The sample size was estimated for the total sample, not for a stratified sample (using the initial BMI such as presented in this article). In this case, an increase in the variability of the small stratum occurs, such as the women classified as low weight at the beginning of prenatal care. At most gestational ages, there were fewer than 10 observations, and in these cases, the rank statistics (such as percentiles) were strongly affected by the sample size (25). These results must be used and evaluated with some restrictions, since a limitation of this study is the altered precision due to the reduced stratified sample size. Another limitation of this study can be considered the statistical method. The approach that was applied was an empirical method followed by a regression analysis to smooth the curves. The absence of dependency of the data between measurements may have resulted in a loss of variance (total and specific models) because we did not consider correlations. However, the number of samples that could be analyzed with dependency was very small due to the lack of data, and the global variance tended to be larger because of the small sample size. Additionally, Atalah’s curve (actual reference curve) was not generated using dependent data (9,25). The curves displayed approximately similar shapes with different levels that were identified when the data were stratified according to the four possible categories of BMI during early prenatal care. Women with low weight early in pregnancy gained weight faster in late pregnancy, as evidenced by the small range of the smoothed percentiles. Women with an adequate initial weight exhibited a uniform BMI increase throughout pregnancy, as demonstrated by the straight and parallel lines with a small range between percentiles at different gestational ages. Other studies have also shown that the differentiation of BMI categories provides important insights into neonatal and maternal outcomes (26,27). The evaluation of curves for overweight women suggests that this group requires special attention because they have a greater range of percentiles during late pregnancy and a steeper slope (sharper increase), which was also observed for underweight women. Overweight women displayed a similar WG to that of women with an adequate WG and a greater WG than that of obese women. Obesity is a known risk factor for adverse outcomes during pregnancy, often representing a bias because obesity may induce clinicians to underestimate the WG problem in overweight and/or adequate-weight women (27). When the IOM guidelines are considered, excessive WG is again highlighted in the overweight group; similar data have also been described in other studies (28,29). These results demonstrate the need for special attention in overweight women. It is natural to anticipate that a standard curve according to the nutritional profile of each woman at the beginning of pregnancy would be feasible and useful. The current proposed model presents BMI percentile curves for women in general and according to BMI at early prenatal care. The results suggest a need for important changes in how BMI and WG are recommended and monitored during pregnancy (26,27). It would be possible and easier to follow the increase in percentiles of BMI similarly as for weight and height increases in children.

’ ACKNOWLEDGMENTS We thank the São Paulo Research Foundation (FAPESP) for providing ﬁnancial support for the current study, grant number 2014/01770-7.

’ AUTHOR CONTRIBUTIONS The idea for the study and this specific analytical approach were developed by Morais SS and Surita FG. Analyses were planned and performed by Morais and Surita FG. The first version of the manuscript was drafted by Morais SS and Surita FG and then complemented with suggestions by Ide M and Morgan AM. Morais SS, Surita FG, Ide M and Morgan AM contributed to the development of the study protocol and approved the final version of the manuscript.

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BASIC RESEARCH

Peri-implant assessment via cone beam computed tomography and digital periapical radiography: an ex vivo study Nicolau Silveira-Neto,I Mateus Ericson Flores,I Joa˜o Paulo De Carli,I Max Do´ria Costa,II Felipe de Souza Matos,III Luiz Renato Paranhos,IV,* Maria Salete Sandini LindenI I Departamento de Odontologia, Universidade de Passo Fundo, Passo Fundo, RS, BR. II Departamento de Odontologia, Universidade Tiradentes, Aracaju, SE, BR. III Departamento de Odontologia Restauradora, Instituto de Cieˆncia e Tecnologia, Universidade Estadual Paulista, Sa˜o Jose´ dos Campos, SP, BR. IV Departamento de Odontologia, Universidade Federal de Sergipe, Lagarto, SE, BR.

OBJECTIVES: This research evaluated detail registration in peri-implant bone using two different cone beam computer tomography systems and a digital periapical radiograph. METHODS: Three different image acquisition protocols were established for each cone beam computer tomography apparatus, and three clinical situations were simulated in an ex vivo fresh pig mandible: buccal bone defect, peri-implant bone defect, and bone contact. Data were subjected to two analyses: quantitative and qualitative. The quantitative analyses involved a comparison of real specimen measures using a digital caliper in three regions of the preserved buccal bone – A, B and E (control group) – to cone beam computer tomography images obtained with different protocols (kp1, kp2, kp3, ip1, ip2, and ip3). In the qualitative analyses, the ability to register peri-implant details via tomography and digital periapical radiography was verified, as indicated by twelve evaluators. Data were analyzed with ANOVA and Tukey’s test (a=0.05). RESULTS: The quantitative assessment showed means statistically equal to those of the control group under the following conditions: buccal bone defect B and E with kp1 and ip1, peri-implant bone defect E with kp2 and kp3, and bone contact A with kp1, kp2, kp3, and ip2. Qualitatively, only bone contacts were significantly different among the assessments, and the p3 results differed from the p1 and p2 results. The other results were statistically equivalent. CONCLUSIONS: The registration of peri-implant details was influenced by the image acquisition protocol, although metal artifacts were produced in all situations. The evaluators preferred the Kodak 9000 3D cone beam computer tomography in most cases. The evaluators identified buccal bone defects better with cone beam computer tomography and identified peri-implant bone defects better with digital periapical radiography. KEYWORDS: Artifacts; Cone Beam Computed Tomography; Dental Implants. Silveira-Neto N, Flores ME, Carli JP, Costa MD, Matos FS, Paranhos LR, et al. Peri-implant assessment via cone beam computed tomography and digital periapical radiography: an ex vivo study. Clinics. 2017;72(11):708-713 Received for publication on June 13, 2017; First review completed on August 9, 2017; Accepted for publication on September 15, 2017 *Corresponding author. E-mail: paranhos@ortodontista.com.br

’ INTRODUCTION

limitation of these methods is the inherent magnification, distortion and lack of three-dimensional information (4). In recent years, cone beam computed tomography (CBCT) has become an important alternative diagnostic tool with high potential for diagnosis and treatment planning, especially for implant treatment (5), by providing three-dimensional images (6,7), which are desirable for determining buccolingual thickness and the morphology and inclination of the alveolar bone (1,4,8). However, if metal, for example, in metal restorations or dental implants, is present in the tomography area, the images tend to display artifacts, which are the main cause of decreased image quality, rendering the images useless for diagnosis in some cases (9,10). Artifacts produced by metal structures, such as titanium dental implants, represent a challenge for automatic processing using computed tomography (CT) scanner software (5,9).

The success of rehabilitation with dental implants requires adequate preoperative planning. Imaging diagnosis and planning methods frequently target the assessment of sites proposed for implant bonding, including the analysis of twodimensional conventional radiographs such as periapical, interproximal, and panoramic radiographs (1-3). The greatest

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CLINICS 2017;72(11):708-713

Figure 1 - (a) Upper view of the planned clinical situations: BBD, PBD, and BC. (b) and (c) Buccal and upper views of the installed implants showing the three clinical situations.

Compared to bone or soft tissue, metallic implants lead to considerable X-ray beam attenuation. This attenuation may produce shadows or beam hardening, which are often obstacles for detailing structures close to these metals, and complicates the postoperative assessment of dental implants with CT (9,11-13). This study aimed to verify detail registration in the periimplant region using CBCT and digital periapical radiograph exams. Three postoperative situations were simulated, and three different image acquisition protocols were used for two CBCT scanners, considering the uncertainty regarding the optimal protocol for tomographic image acquisitions with respect to the reduction of metal artifacts and higher registration accuracy in clinical situations.

Table 1 - Specifications for the Kodak 9000 3D CBCT. Parameters kVp mAs Voxel FOV (mm) Exam time (s) Software

kp1

kp2

kp3

68 8.0 0.076 4.7 x 5.9 10.8 Kodak Dental Imaging Software 3D

60 6.3 0.1 4.7 x 5.9 10.8 Kodak Dental Imaging Software 3D

80 12.0 0.2 4.7 x 5.9 10.8 Kodak Dental Imaging Software 3D

Table 2 - Specifications for the i-CAT CBCT. Parameters

’ MATERIALS AND METHODS

kVp mAs Voxel FOV (mm) Exam time (s) Software

Production of an Experimental Model

In a fresh pig mandible (ex vivo), an installation was planned with three cylindrical, external hexagonal titanium dental implants that were 3.75 x 15 mm in size (Conexão Sistemas de Prótese, São Paulo, SP, Brazil) and spaced at least 5 mm apart using a surgical guide previously made with a casting and mandible plaster model. The perforations for implant installations simulated an in vivo installation according to the manufacturer’s instructions for working with medullary bone, with purposeful under instrumentation to produce the highest bone contact with the implants. A motor (Driller BLM 600 Plus, Driller, São Paulo, SP, Brazil) and a 20:1 surgical contra-angle handpiece (W&H Dentalmechanik, Bürmos, Austria) were used at 1000 rpm under constant saline solution irrigation. The burs used were P-i Branemarkt diamond-like carbon (Exopro, São Paulo, SP, Brazil). Before implant installation, the cervical regions were assessed to produce three clinical situations (Figure 1): buccal bone defect (BBD), which was simulated by buccal wear with a KG Sorensen 3215 diamond bur (KG Sorensen, Cotia, SP, Brazil) under high rotation using a Kavo Extra Torque 650C (Kavo, Joinville, SC, Brazil); peri-implant bone defect (PBD), which was simulated by enlargement of the surgical cavity with a 5-mm-diameter Dense Drill from P-i Branemarkt (Exopro, São Paulo, SP, Brazil), preserving 4 mm of bone contact in the apical region for implant stability at the moment of installation; and bone contact (BC), which was a full bone overlay with no bone defect simulation. Next, implants were installed according to the manufacturer’s instructions. Initially, an internal grip driver from P-i Branemarkt (Exopro, São Paulo, SP, Brazil) was used with a motor at 26 rpm, and a manual torque wrench from P-i Branemarkt (Exopro, São Paulo, SP, Brazil) was used to complete the insertion.

ip1

ip2

ip3

120 37.07 0.2 8.0 x 8.0 26.09 i-CAT Vision Xoran

120 20.27 0.125 8.0 x 8.0 26.09 i-CAT Vision Xoran

120 18.54 0.4 8.0 x 8.0 8.9 i-CAT Vision Xoran

Image Acquisition To test the hypothesis that detail registration in peri-implant regions is influenced by the image acquisition protocol used, two CBCT scanners were used: a Kodak 9000 3D CBCT (Dental Systems, Carestream Health, Rochester, USA) and an i-CAT CBCT (Imaging Sciences International, Hatfield, USA). Three image acquisition protocols were used for each apparatus (Tables 1 and 2). A container with the experimental model was placed on the exam platform for each CT scanner and was fixed and aligned using guidance lasers. After CT exams, the software of each scanner standardized the transverse obliquesectioned images and the most central specimen section of each implant. The images were organized in three templates corresponding to each postoperative situation (BBD, PBD, and BC) (Figure 2). A periapical radiograph was also taken using a chargecoupled device (CCD) digital sensor of the Kodak RVG 6100 (Kodak Dental System) model. Each implant was radiographed at an orthoradial angle with the parallelism technique following the geometric principles of radiographic image formation. After image acquisition, the specimen was sectioned in a cutting machine (Miniton, Struers, Copenhagen, Denmark) with a diamond disc at the central point of the implants and parallel to the long axis in order to match the images previously selected in the CT scanner software. After sectioning, the

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Figure 2 - Image results from the BBD, PBD, and BC cases for the different image acquisition protocols from sagittal CBCT images.

Figure 3 - Measurements of a real object made with a digital caliper in regions A, B, and E.

was performed by twelve dentists experienced in implantology or dental radiology and in the routine use of CT images in order to verify which CT scanners and acquisition protocols were able to more accurately register the study object. The dentists performed an isolated and blind assessment, as they were not informed as to which CT scanner produced the image shown. During the assessment, the evaluator was presented with a photograph of the transverse oblique section of the real object, images corresponding to each CT scanner and protocol used, a photograph of the buccal aspect of the specimen before sectioning, and periapical radiographs of each implant (Figure 4). Each dentist was asked to rate the ability of the tomographic image to accurately register peri-implant details in each situation on a scale (14) from 1 to 5, with the following score options: 1 - very poor, 2 - poor, 3 - good, 4 - very good, 5 - excellent. The data were analyzed with ANOVA and Tukey’s test (a=0.05).

portions chosen for implant assessment were photographed with a digital camera (Nikon D80, Nikon, Japan) using a macro lens and a Sigma circular flash for Nikon (Sigma, Japan) for qualitative analysis. Moreover, a properly calibrated examiner measured the real object using a digital caliper (Cosa Vinhedo, SP, Brazil) in three regions of the preserved buccal portion (Figure 3): region A corresponded to the vertical measure (height) of the prosthetic platform of the implant up to the first buccal bone contact to the implant, region B was the bone thickness measure of the first buccal bone contact to the implant, and region E indicated the buccal bone thickness measure in the middle third of the implant. These measurements were repeated five times for each region at different times, and the following control (CG) measures were considered: CBBD-A, CPBD-A, and CBC-A - vertical buccal measures for region A in each implant, CBBD-B, CPBD-B, and CBC-B - horizontal buccal measures for region B in each implant, CBBD-E, CPBD-E, and CBC-E - horizontal buccal measures for region E in each implant. The same measurements were also taken in these regions using the images obtained from each CT scanner software program with the different image acquisition protocols. The images were assessed by two experienced and trained dentists for each scanner, one dentist for each scanner studied. Three measurements were taken at different times for each region.

’ RESULTS Quantitative Analysis Tables 3 and 4 present the mean values for the different image acquisition protocols (Kodak - kp1, kp2, kp3 and i-CAT - ip1, ip2, ip3) as well as the true values found by direct measurement (CG) in the three regions measured (region A, B, and E) for the three simulated situations (BBD, PBD, and BC).

Data Analysis All quantitative data were tabulated and analyzed with ANOVA and Tukey’s test (a=0.05). Qualitative assessment

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Figure 4 - Model of presentation for evaluators for the PBD situation.

Table 3 - Mean values for the BBD, PBD, and BC cases using the Kodak 9000 3D CBCT. Protocol

Region A (mm)

BBD - buccal bone defect Control 4.622b kp1 4.730a kp2 4.686a kp3 4.690a

Region B (mm)

Region E (mm)

0.688a 0.704a 0.528b 0.534b

2.750a 2.740a 2.640b 2.604b

Table 5 - Score interpretations for tomographic images and digital periapical radiographs in each situation. Situation

BBD

PBD PBD - peri-implant bone defect Control 9.914a kp1 8.980c kp2 9.676b kp3 9.560b BC - bone contact Control 0.878a kp1 0.914a kp2 0.918a kp3 0.872a

3.216 2.856c 3.040b 2.914c

0.862 0.700b 0.574c 0.588c

2.858 2.784b 2.846a 2.810ab

2.518 2.450b 2.408bc 2.370c

Region B (mm)

Region E (mm)

BBD - buccal bone defect Control 4.622a ip1 4.234d ip2 4.500b ip3 4.384c

0.688a 0.620ab 0.570b 0.394c

2.750a 2.260c 2.510b 2.020d

PBD - peri-implant bone defect Control 9.914a ip1 8.712c ip2 8.980b ip3 8.888bc

3.216a 2.592b 2.586b 2.460c

2.858a 2.632b 2.510c 2.010d

BC - bone contact Control 0.878a ip1 0.648c ip2 0.856a ip3 0.764b

0.862a 0.534c 0.564c 0.724b

2.518a 2.388b 2.250c 1.736d

p1 - Very Good p2 - Very Good p3 - Very Good p1 - Very Poor p2 - Very Poor p3- Very Poor p1- Very Good p2 - Very Good p3- Poor

p1- Good p2 - Good p3 - Good p1 - Poor p2 - Very Poor p3 - Very Poor p1 - Poor p2 - Good p3 - Poor

Digital Periapical Radiograph

Very Poor

Very Good

’ DISCUSSION

Table 4 - Mean values for the BBD, PBD, and BC cases using the i-CAT CBCT. Region A (mm)

i-CAT

scanners; only the BC situation gave significantly different results. The results for acquisition protocol 3 differed from those for 1 and 2. The results for the other protocols were not significantly different (Table 5). The interpretation of scores for tomographic images and digital periapical radiographs in each situation in terms of the ability of each image to register the simulated bone condition is shown in Table 5.

Means followed by the same letter are not significantly different (Tukey’s test, a=0.05).

Protocol

Kodak

CT is an important imaging method for the diagnosis of lesions in both hard and soft tissues of the oral cavity and the head and neck region. In recent years, CBCT has become an important alternative diagnostic tool due to its high potential for diagnosis and treatment planning, especially for implant treatment, by providing three-dimensional images (1,5-8). However, artifacts cause decreased image quality in CBCT, which in some cases may render such images useless for diagnosis (9,10). An artifact is any type of image distortion or error that is not related to the object of study. Artifacts produced by metal structures, such as titanium dental implants, represent a challenge for automatic processing using CT scanner software (5,9). Schulze et al (15) assessed the image quality of the New Tom 900 and Siemens Siremobil in a dehydrated skull and reported no artifact formation, which was attributed to the fact that no metal structures were used in the study. The present work observed different quantities of metal artifacts in all exams due to the presence of titanium implants; compared to bone or soft tissue, titanium implants cause considerable X-ray beam attenuation. This attenuation may produce shadows or beam hardening, which are often obstacles for detailing structures close to these metals, complicating the postoperative assessment of dental implants with CT (9-13).

Means followed by the same letter are not significantly different (Tukey’s test, a=0.05).

Qualitative Analysis According to the scores assigned by the evaluators, similar performances were verified for the images from both CT

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the formation of artifacts and accurately portray the real condition, which suggests, for some situations examined such as kp1, kp2, kp3, and ip3, a completely non-existent bone contact. The difficulty in defining the region of bone contact with the implants was unanimous for both radiologists and implantologists, which agrees with reports by Ravazi et al (12) and Schulze et al (13). Some studies have investigated the difference in diagnostic accuracy between CBCT and periapical radiography (1-3,8,16). The majority revealed the superiority of CBCT to periapical radiography for detecting apical lesions. A similar result was found by Estrela et al (16), who found 100% sensitivity in detecting lesions with CBCT and a much lower sensitivity with panoramic (28%) and periapical (55%) radiography. In the present research, the digital periapical radiograph was preferred among evaluators for detecting the simulated PBD, which may be attributed to the absence of artifacts. Due to structural overlaps, which are characteristic of periapical radiographs, the evaluators identified the BBD case better using CBCT. Considering the relevance of the present study in clarifying diagnostic imaging methods in patients with dental implants, it should be highlighted that the experiment was performed in an ex vivo model of a dry swine jaw. This approach differs from the methodology used in previous studies (17,18), presenting a limitation of the present study due to the absence of soft tissue during image acquisition. The registration of peri-implant details is influenced by the image acquisition protocol, although metal artifacts were produced in all situations. The Kodak 9000 3D CBCT registered the study object more accurately than the i-CAT CBCT. The evaluators preferred digital periapical radiography for detecting the simulated peri-implantitis, while the buccal bone defect was identified better using CBCT.

The use of higher kVp (kp3 - Kodak 9000 3D CBCT) and mAs (ip1 - i-CAT CBCT) values allowed for images with measures statistically equal to those of the CG only in the kp3-PBD region E, kp3-BC region A, and ip1-BBD region B situations. Kataoka et al (11) suggested that an increase in kVp and mAs may help control metal artifact formation. Esmaeili et al (10) also reported that the high resolution of images produced by the New Tom VGi and Somatom Sensation may be attributed to the high kVp used for image acquisition. Corroborating these authors, Chindasombatjareon et al (6), when assessing the production of metal artifacts in the Light Speed QX/I CBCT and Alpha Veja 3030 CBCT systems, observed that an increase in kVp in both CT scanners led to a reduction in artifacts. In contrast, the use of lower kVp (kp1 - Kodak 9000 3D CBCT) and mAs (ip3 - i-CAT CBCT) values also resulted in images with measures statistically equal to the those of the CG in the kp1-BBD region B, kp1-BBD region E, and kp1-BC region A situations. Shulze et al (13), in a study performed using a phantom with CBCT, found similar results and affirmed that using a lower kVp (80 compared to 90 and 120) resulted in reduced beam hardening. Some intermediate values of kVp showed irregularities in results when the voxel size (kp2 - Kodak 9000 3D CBCT) and mAs (ip2 - i-CAT CBCT) were reduced, producing images with measures statistically equal to those of the CG in the kp2-PBD region E, kp2-BC region A, and ip2-BC region A situations. Similarly, Ravazi et al (12), in a study performed on a bovine rib with i-CAT NG (120 kV, 18.54 mAs, voxel 0.3, FOV 8x16 cm) and Accuitomo 3D60 FPD (80 kVp, 4 mAs, voxel 0.125 mm, FOV 6x6 cm), verified that both instruments overestimated the distance from the implant vertical to the bone crest, with better images obtained by the Accuitomo 3D60 FPD. The present study also showed that the ip3 acquisition protocol had the most disparate results from the CG. This result may be explained by the larger voxel size and lower mAs among the protocols used for i-CAT CBCT. All protocols used for the Kodak 9000 3D CBCT had lower kVp and mAs values than those for the i-CAT CBCT. There was a variation between underestimation and overestimation of screw display and bone thickness analyzed, with some cases suggesting a higher bone loss than that found in the real object. Other cases suggested the presence of bone tissue in places not found in the real object. The kp1-BBD region A, kp2-BBD region A, and kp3-BBD region A situations showed higher average measures than those of the CG. In contrast, the protocols set for i-CAT had an overall tendency to reduce the average measures relative to the CG. The qualitative assessment indicated a superiority of the Kodak 9000 3D CBCT for BBD and BC situations because, according to the evaluators, artifact formation did not compromise the identification of peri-implant characteristics in these situations. For the PBD situation, both CBCT apparatuses obtained intermediate scores, with i-CAT obtaining a higher score, which may be attributed to the use of higher kVp and mAs values in the exam acquisitions, preventing masking of the bone defect simulated around the implant by artifacts. Statistically, according to the scores assigned by the evaluators, only the BC situation produced different results, where the p3 results were different from the p1 and p2 results. The dentists involved in the qualitative assessment were surprised, especially when observing the images simulated for the PBD case, that no acquisition protocol could reduce

â&#x20AC;&#x2122; AUTHOR CONTRIBUTIONS Flores ME and Silveira-Neto N were responsible for the conception and design of the study. Flores ME and Linden MS were responsible for project coordination. Matos FS and Paranhos LR drafted the manuscript. Flores ME and Silveira-Neto N were responsible for the data collection. Carli JP and Paranhos LR were responsible for the statistical analyses. Silveira-Neto N, Paranhos LR, Matos FS, Costa MD, Flores ME, Carli JP and Linden MS edited and revised the manuscript.

â&#x20AC;&#x2122; REFERENCES 1. Dave M, Davies J, Wilson R, Palmer R. A comparison of cone beam computed tomography and conventional periapical radiography at detecting peri-implant bone defects. Clin Oral Implants Res. 2013;24(6): 671-8, http://dx.doi.org/10.1111/j.1600-0501.2012.02473.x. 2. Ritter L, Elger MC, Rothamel D, Fienitz T, Zinser M, Schwarz F, et al. Accuracy of peri-implant bone evaluation using cone beam CT, digital intra-oral radiographs and histology. Dentomaxillofac Radiol. 2014; 43(6):20130088, http://dx.doi.org/10.1259/dmfr.20130088. 3. Bagis N, Kolsuz ME, Kursun S, Orhan K. Comparison of intraoral radiography and cone-beam computed tomography for the detection of periodontal defects: an in vitro study. BMC Oral Health. 2015;28; 15-64, http://dx.doi.org/10.1186/s12903-015-0046-2. 4. Scarfe W, Farman AG. Interpreting CBCT images for implant assessment: Part 1 - Pitfalls in image interpretation. Aust Dent Pract. 2010;20:106-14. 5. Yepes JF, Al-Sabbagh M. Use of cone-beam computed tomography in early detection of implant failure. Dent Clin North Am. 2015;59(1):41-56, http://dx.doi.org/10.1016/j.cden.2014.09.003. 6. Chindasombatjareon J, Kakimoto N, Murakami S, Maeda Y, Furukawa S. Quantitative analysis of metallic artifacts caused by dental metals: comparison of cone-beam and multi-detector row CT scanners. Oral Radiol. 2011;27(2):114-20, http://dx.doi.org/10.1007/s11282-011-0071-z.

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13. Schulze RKW, Berndt D, Dâ&#x20AC;&#x2122;Hoedt B. On cone-beam computed tomography artifacts induced by titanium implants. Clin Oral Implants Res. 2010;21(1):100-7, http://dx.doi.org/10.1111/j.1600-0501.2009.01817.x. 14. Likert R. "A technique for the measurement of attitudes". Arch Psychol. 1932;22(140):1-55. 15. Schulze D, Heiland M, Blake F, Rother U, Schmelzle R. Evaluation of quality of reformatted images from two cone-beam computed tomographic systems. J Craniomaxillofac Surg. 2005;33(1):19-23, http://dx.doi. org/10.1016/j.jcms.2004.07.004. 16. Estrela C, Bueno MR, Leles CR, Azevedo B, Azevedo JR. Accuracy of cone beam computed tomography and panoramic and periapical radiography for detection of apical periodontitis. J Endod. 2008;34(3):273-9, http://dx. doi.org/10.1016/j.joen.2007.11.023. 17. Corpas Ldos S, Jacobs R, Quirynen M, Huang Y, Naert I, Duyck J. Periimplant bone tissue assessment by comparing the outcome of intra-oral radiograph and cone beam computed tomography analyses to the histological standard. Clin Oral Implants Res. 2011;22(5):492-9, http://dx. doi.org/10.1111/j.1600-0501.2010.02029.x. 18. Lau SL, Chow LK, Leung YY. A non-invasive and accurate measurement of gingival thickness using cone-beam computerized imaging for the assessment of planning immediate implant in the esthetic zone-a pig jaw model. Implant Dent. 2016;25(5):619-23, http://dx.doi.org/10.1097/ ID.0000000000000437.

7. Scarfe WC, Farman AG, Sukovic P. Clinical applications of cone-beam computed tomography in dental practice. J Can Dent Assoc. 2006;72(1): 75-80. 8. Fienitz T, Schwarz F, Ritter L, Dreiseidler T, Becker J, Rothamel D. Accuracy of cone beam computed tomography in assessing peri-implant bone defect regeneration: a histologically controlled study in dogs. Clin Oral Implants Res. 2012;23(7):882-7, http://dx.doi.org/10.1111/j.1600-0501.2011.02232.x. 9. Kamburoglu K, Kolsuz E, Murat S, Eren H, YĂźksel S, Paksoy CS. Assessment of buccal marginal alveolar peri-implant and periodontal defects using a cone beam CT system with and without the application of metal artefact reduction mode. Dentomaxillofac Radiol. 2013;42(8): 20130176, http://dx.doi.org/10.1259/dmfr.20130176. 10. Esmaeili F, Johari M, Haddadi P. Beam hardening artifacts by dental implants: comparison of cone-beam and 64-slice computed tomography scanners. Dent Res J. 2013;10(3):376-81. 11. Kataoka ML, Hochman MG, Rodriguez EK, Lin PJ, Kubo S, Raptopolous VD. A review of factors that affect artifact from metallic hardware on multi-row detector computed tomography. Curr Probl Diagn Radiol. 2010;39(4):125-36, http://dx.doi.org/10.1067/j.cpradiol.2009.05.002. 12. Razavi T, Palmer RM, Davies J, Wilson R, Palmer PJ. Accuracy of measuring the cortical bone thickness adjacent to dental implants using cone beam computed tomography. Clin Oral Implants Res. 2010;21(7):718-25, http://dx.doi.org/10.1111/j.1600-0501.2009.01905.x.

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RAPID COMMUNICATION

Evidence of progenitor cells in the adult human cochlea: sphere formation and identification of ABCG2 Milene Massucci-Bissoli, * Karina Lezirovitz, * Jeanne Oiticica, Ricardo Ferreira Bento Departamento de Otorrinolaringologia, Faculdade de Medicina FMUSP, Universidade de Sao Paulo, Sao Paulo, SP, BR.

OBJECTIVES: The aim of this study was to search for evidence of stem or progenitor cells in the adult human cochlea by testing for sphere formation capacity and the presence of the stem cell marker ABCG2. METHODS: Cochleas removed from patients undergoing vestibular schwannoma resection (n=2) and from brain-dead organ donors (n=4) were dissociated for either flow cytometry analysis for the stem cell marker ABCG2 or a sphere formation assay that is widely used to test the sphere-forming capacity of cells from mouse inner ear tissue. RESULTS: Spheres were identified after 2-5 days in vitro, and the stem cell marker ABCG2 was detected using flow cytometric analysis after cochlear dissociation. CONCLUSIONS: Evidence suggests that there may be progenitor cells in the adult human cochlea, although further studies are required. KEYWORDS: Progenitor Cells; Cochlea; ABCG2; Human. Bissoli MM, Lezirovitz K, Oiticica J, Bento RF. Evidence of progenitor cells in the adult human cochlea: sphere formation and identification of ABCG2. Clinics. 2017;72(11):714-717 Received for publication on March 2, 2017; First review completed on May 31, 2017; Accepted for publication on July 12, 2017 *Corresponding authors. E-mails: milene.bissoli@gmail.com / lezi.karina@gmail.com

not differentiate in vitro and exhibited few developmental markers (10). In neonatal mice, there also exists evidence of a side population of cochlear cells that express the stem cell marker ABCG2 and are capable of in vitro proliferation, self-renovation and differentiation (11-14). The aim of this study was to identify evidence of progenitor or stem cells in the adult human cochlea. To test our hypothesis, we used cochleas removed from patients undergoing vestibular schwannoma (VS) resection and brain-dead organ donors; this article is the first report involving the use of cochlear tissue from organ donors.

’ INTRODUCTION Hearing loss is one of the most common sensorial deficiencies in humans. Although there exist several options for treating hearing loss, none of these approaches can completely reestablish auditory physiology. Prosthetic devices require maintenance and have inherent daily limitations due to their electrical nature; many such devices are simply not designed to be used while sleeping. One possible strategy to reestablish auditory physiology is the replacement of lost sensorial cells. In theory, the discovery of adult stem cells in the cochlea would bring this possibility closer to clinical practice. No prior studies have attempted to find evidence of stem cells in the adult human cochlea. The availability of human cochlear tissue is limited, and most authors have chosen to use cochleas from patients who are undergoing surgical procedures that suggest permanent hearing loss (1-4). The only references to human cochlear stem cells involve fetal specimens (5-6). Sphere formation assays have been used to demonstrate the proliferative capacity of neonatal mammalian cochleas in different species (7-9). Spheres have also been identified in adult mice, although such spheres were not numerous, could

’ MATERIALS AND METHODS Patients Cochlear samples were obtained from patients undergoing excision of a VS via a translabyrinthine approach (n=2) and brain-dead organ donors (n=4) at University of Sao Paulo Clinics Hospital. All patients or their legal representatives provided informed consent prior to tissue collection in accordance with requirements of local and national ethics committees.

Surgery in the VS group A translabyrinthine approach was utilized by the same surgeon for both patients, and cochlear access was achieved as described by Browne and Fisch (15). After cochlear exposure, a wide cochleotomy was performed to allow for tissue removal. The membranous portion of the cochlea was collected into culture medium for immediate transport to the laboratory.

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Tissue collection in the organ donor group

Eagle’s minimum essential medium (EMEM; Vitrocell Embriolife, Campinas, SP, Brazil). Due to limited equipment availability, samples remained at 4oC for up to 36 hours; they were then dissociated with trypsin and mechanically dissociated (16) for antibody incubation in accordance with the manufacturer’s instructions. We used an anti-ABCG2 antibody (Biolegend, 332020, San Diego, CA, USA) and its isotype (Biolegend, 401209), and flow cytometric analysis was performed on an Attunes NxT Acoustic Focusing Cytometer (Thermo Fisher Scientific, Inc., Waltham, MA, USA).

To reach the cochlear promontory, three different approaches were tested. On one side, we used an endaural approach; on the remaining sides, retroauricular access was established to perform a mastoidectomy. On four sides, a posterior tympanotomy was conducted to gain access to the cochlear promontory. On one side, the posterior wall of the external auditory canal was drilled away to improve cochlear visualization. In all cases, after cochlear exposure, a wide cochleotomy was performed to allow for tissue removal. The membranous portion of the cochlea was collected into culture medium for immediate transport to the laboratory.

’ RESULTS

Sphere formation assay

Patients and tissue collection

To test the ability of removed cochlear tissue to form spheres, we used the protocol described by Oshima et al. (16). Briefly, tissue was inspected with an inverted microscope, dissociated with trypsin, mechanically passed through a 70 mm filter and cultured under non-adherent conditions on defined media (DMEM-F12 with 2 B-27, 1 N2, 2 mM glutamine, 2 mL/mL ITS, 6 g/L glucose, 0.2 mL/mL ampicillin, 20 ng/mL EGF, 10 ng/mL bFGF and 50 ng/mL IGF) for up to five days.

The patients in the VS group (both female) were 61 and 63 years of age, and those in the organ donor group (2 males and 2 females) were 17-55 years of age. Both patients had moderate unilateral sensorineural hearing loss. In the organ donor group, six of the eight available sides were used. We have no auditory testing data from this group. There was great variability in the quantities of tissue collected from patients and organ donors. In all cases, a portion of the organ of Corti and stria vascularis could be identified. In the organ donor group, the greatest quantity of tissue was collected when we used a retroauricular approach followed by mastoidectomy with drilling of the posterior wall of the external auditory canal. In the VS group, variability in tissue quantities was mainly attributable to anatomical variations. Figure 1 depicts a section of the membranous portion of the cochlea shortly after its removal from a patient undergoing VS resection.

Flow cytometry To verify the presence of the stem cell marker ABCG2, removed cochlear tissue was transferred to the laboratory in

Identification of spheres Three independent sphere formation assays were performed. Two of these assays used samples from the VS group (involving one cochlea each), and the remaining assay utilized a sample from the organ donor group (with two cochleas assessed in the same experiment). The evaluated cochleas were from 61- and 63-year-old female patients who were undergoing surgery and a 17-year-old male organ donor. Spheres were identified in two of the three experiments (Figure 2).

Identification of ABCG2 Four cochleas from three organ donors were used for flow cytometric identification of ABCG2. The donors included one 38-year-old male and 33- and 55-year-old females. There was a clear population of ABCG2-positive

Figure 1 - Membranous portion of the cochlea that was removed from a patient undergoing VS resection.

Figure 2 - Sphere identification for samples from 2A, a 61-year-old female undergoing VS resection at 5 DIV; 2B, a 17-year-old male organ donor at 3 DIV; and 2C, a 17-year-old male organ donor at 5 DIV.

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Figure 3 - Flow cytometric analysis of a dissociated cochlea from a 33-year-old female (3B). There exists a population of cells positive for ABCG2 (R2) that represents 1.04% of the total cell population (3A).

cochleas from brain-dead organ donors, the standardization of the procedure for membranous cochlear resection, and the development of greater expertise with this procedure. Variability and little reproducibility in the removal of membranous cochlear tissue have been reported previously (1). However, with more widespread use of this material, this issue can be solved. We have demonstrated evidence of the presence of progenitor cells in the adult human cochlea in the form of sphere formation and the detection of ABCG2. These findings do not conclusively establish the presence of stem cells in this tissue, but they do open a new field for stem cell research. We have reported the first use of brain-dead organ donors for cochlear resection for research purposes; this approach can be of inestimable value for hearing research.

cells in the dissociated cochlea from the 33-year-old female (Figure 3).

Comparative table of results Age

Gender

Experiment

Result

61 63 17 38 55 33

female female male male female female

sphere formation assay sphere formation assay sphere formation assay ABCG2 identification ABCG2 identification ABCG2 identification

negative positive positive inconclusive inconclusive positive

’ DISCUSSION

’ ACKNOWLEDGMENTS

This article is the first report describing sphere formation and the identification of ABCG2 in the adult human cochlea and the first investigation in which brain-dead organ donors were a source of cochleas used for research. The accurate identification of stem cells in vivo remains a major obstacle to understanding in stem cell biology since there are no single and universal stem cell markers common to all adult stem cells (17). To identify evidence of stem or progenitor cells in the adult human cochlea, we used a sphere-forming assay validated for neonatal mice (16) that has been used to test the sphereforming capacity of cells from adult mice (10). We identified spheres in two out of three experiments; nevertheless, the number of spheres was minimal, and no further testing could be adequately performed for further characterization. Prior research has demonstrated that neonatal mice have a cochlear side population that expresses the stem cell marker ABCG2 (11). Several authors have established that the direct identification of ABCG2 in this tissue is a marker of this side population (18-20). We have used flow cytometric analysis to reveal the presence of ABCG2 in adult human dissociated cochleas. We have been unable to sort and regrow ABCG2positive populations due to the small number of cells obtained in our experiments, but testing to determine whether these cells behave similarly to mouse cochlear side population/ ABCG2-positive cells would be extremely valuable (12-14). A greater number of cochlear cells could be obtained for the aforementioned experiments via the collection of more

We thank Dr. Tharcísio Citrângulo Tortelli, Junior, for helping with flow cytometry analysis and doctors Fábio de Alencar Rodrigues, Junior, Francisco das Chagas Cabral, Junior, Paula Tardim Lopes and Ricardo Dourado Alves for performing the organ donor cochlear resections. Funding: This work was supported by the Brazilian National Council of Technological and Scientific Development (CNPq) in the form of a scholarship to the first author.

’ AUTHOR CONTRIBUTIONS Bissoli MM was responsible for the manuscript preparation, cochlear preparation after its removal from patients and cell culture and staining for ﬂow cytometry. Lezirovitz K was responsible for co-mentoring, manuscript preparation and ﬁgure selection. Oiticica J was responsible for the cochlear preparation after its removal from patients in selected cases and mentoring. Bento RF was responsible for the surgery, mentoring and manuscript preparation.

’ REFERENCES 1. Oghalai J, Holt JR, Nakagawa T, Jung TM, Coker NJ, Jenkins HA, et al. Harvesting human hair cells. Ann Otol Rhinol Laryngol. 2000;109(1):9-16, http://dx.doi.org/10.1177/000348940010900102. 2. Rask-Andersen H, Boström M, Gerdin B, Kinnefors A, Nyberg G, Engstrand T, et al. Regeneration of human auditory nerve. In vitro/ in video demonstration of neural progenitor cells in adult human and guinea pig spiral ganglion. Hear Res. 2005;203(1-2):180-91, http://dx.doi. org/10.1016/j.heares.2004.12.005. 3. Wei L, Rui G, Helge RA. Morphological Study of Surgically Obtained Human Cochlear Specimens – Technical Aspects. Journal of Otology. 2014;9(1):52-7, http://dx.doi.org/10.1016/S1672-2930(14)50010-4.

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Evidence of progenitor cells in adult human cochlea Massucci-Bissoli M et al.

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Seja pequeno, médio ou grande, o Rebouças tem o espaço ideal para todo o tipo de evento. A melhor logística e infraestrutura de apoio maximizam os recursos operacionais e potencializam os encontros científicos, promocionais, institucionais ou culturais. O Centro de Convenções Rebouças é isso: um dos mais bem planejados e tradicionais espaços receptivos do País, dimensionado para sediar congressos, convenções, exposições, seminários, simpósios, cursos, lançamento de produtos, entre outros, bem no coração da cidade de São Paulo.

Atualmente possui 8 ambientes climatizados e modernizados que atendem até 1.200 participantes, além de uma equipe preparada e empenhada em acompanhar cerca de 280 eventos por ano. EM BREVE, estará ampliando suas instalações, que permitirá o dobro da capacidade de público.

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