December 3 - Volume Number 6 ISSN Galeria do Rock Mercedes Lorenzo Finotti / istock.com

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1 ISSN São Paulo Medical Journal/Evidence for Health Care, 2015 December 3; 133(6): S Ã O P A U L O E V I D E N C E F O R H E A L T H C A R E December 3 - Volume Number 6 Cross-sectional accuracy study: Assessment of a glycated hemoglobin point-ofcare analyzer (A1CNow+) in comparison with an immunoturbidimetric method: a diagnostic accuracy study Cross-sectional study: Elevated gamma glutamyl transferase levels are associated with the location of acute pulmonary embolism Prospective cohort study: Cohort study on the factors associated with survival postcardiac arrest Medline, Lilacs, SciELO, Science Citation Index Expanded, Journal Citation Reports/ Sciences Edition (impact factor 0.723) and EBSCO Publishing Galeria do Rock Mercedes Lorenzo Finotti / istock.com Federada da São Paulo Medical Journal does not charge authors for publication.

2 23 anos de Precisão, Qualidade e Confiança ligue e confira (11) Mudanças Residenciais Mudanças Empresariais Equipamentos sensíveis (informática, robótica e hospitalares) Embalagens Especiais (obras de arte, exposições e antiguidades) 20% de desconto Remoções Internas (confecção de lay-out), Içamentos em todas as modalidades Guarda-móveis (armazenagem). Rua Doze de Setembro, 856 mega@megamudancas.com.br

3 INDEX Correspondence to: ASSOCIAÇÃO PAULISTA DE MEDICINA Publicações Científicas Av. Brig. Luís Antônio, o andar São Paulo (SP) Brasil CEP Tel. (+55 11) ou (+55 11) Fax: (+55 11) revistas@apm.org.br APOIO Editorial 457 Stroke is still a neglected disease in Brazil Paulo Andrade Lotufo Original article 460 Assessment of a glycated hemoglobin point-of-care analyzer (A1CNow+) in comparison with an immunoturbidimetric method: a diagnostic accuracy study Aurélie Affret, Luiz Henrique Maciel Griz, Eduarda Ângela Pessoa Cesse, Yuri da Silva Specht, Eduardo Maia Freese de Carvalho, Annick Fontbonne 465 Bacterial vaginosis in pregnant adolescents: proinflammatory cytokine and bacterial sialidase profile. Cross-sectional study Carolina Sanitá Tafner Ferreira, Camila Marconi, Cristina Maria de Lima Garcia Parada, Marli Teresinha Cassamassimo Duarte, Ana Paula Oliveira Gonçalves, Marilza Vieira Cunha Rudge, Márcia Guimarães da Silva 471 Patients perceptions about diagnosis and treatment of chronic myeloid leukemia: a cross-sectional study among Brazilian patients Nelson Hamerschlak, Carmino de Souza, Ana Lúcia Cornacchioni, Ricardo Pasquini, Daniel Tabak, Nelson Spector, Merula Steagall 480 Clinical management of the first ASCUS report in Chile. Prospective single-cohort study Fanny López-Alegría, Orlando Quezada Poblete, Dino Soares De Lorenzi, Juan Carlos Sepúlveda Oyanedel 488 Elevated gamma glutamyl transferase levels are associated with the location of acute pulmonary embolism. Cross-sectional evaluation in hospital setting Ozge Korkmaz, Hasan Yucel, Ali Zorlu, Ocal Berkan, Hakki Kaya, Sebahattin Goksel, Osman Beton, Mehmet Birhan Yilmaz 495 Cohort study on the factors associated with survival post-cardiac arrest Cássia Regina Vancini-Campanharo, Rodrigo Luiz Vancini, Claudio Andre Barbosa de Lira, Marília dos Santos Andrade, Aécio Flávio Teixeira de Góis, Álvaro Nagib Atallah 502 Attributable causes of chronic kidney disease in adults: a five-year retrospective study in a tertiary-care hospital in the northeast of the Malaysian Peninsula Muhammad Salman, Amer Hayat Khan, Azreen Syazril Adnan, Syed Azhar Syed Sulaiman, Khalid Hussain, Naureen Shehzadi, Fauziah Jummaat 510 Sodium and potassium intake estimated using two methods in the Brazilian Longitudinal Study of Adult Health (ELSA-Brasil) Taísa Sabrina Silva Pereira, Isabela Judith Martins Benseñor, Jorge Gustavo Velásquez Meléndez, Carolina Perim de Faria, Nágela Valadão Cade, José Geraldo Mill, Maria del Carmen Bisi Molina Short communication 517 Urinary tract infection and indwelling urinary catheters: prospective study in gynecological surgery with antibiotic prophylaxis José Carlos Carraro-Eduardo, Daniela da Silva Alves, Ingrid Ellis Hinden, Ivan Penaloza Toledano, Sarah Gomes Freitas, Pedro Juan José Mondino, José Rodrigo de Moraes, Carlos Augusto Faria Case series 521 Down syndrome: Prevalence and distribution of congenital heart disease in Brazil Beatriz Elizabeth Bagatin Veleda Bermudez, Sandra Lira Medeiros, Mariane Bagatin Bermudez, Iolanda Maria Novadzki, Neiva Isabel Rodrigues Magdalena Case report 525 Liver transplantation in a patient with hepatitis B, C and D coinfection associated with hepatocellular carcinoma: a management strategy for a rare condition. Case report Lucas Carvalho Dantas, Tércio Genzini, Marcelo Perosa de Miranda, Regina Gomes dos Santos, Nilton Ghiotti de Siqueira, Judith Weirich, Cirley Maria de Oliveira Lobato 531 Removing a broken guidewire in the hip joint: treatment options and recommendations for preventing an avoidable surgical catastrophe. A case report Abhijeet Ashok Salunke, Prem Haridas Menon, Gurunathampalayam Ilango Nambi, Junhao Tan, Vivek Patel, Yongsheng Chen, Jay Kumar Letter to the editor 535 Personality disorders in medical psychology: medical training and professional creativity Décio Gilberto Natrielli Filho, Décio Gilberto Natrielli, Anderson Souza Martins da Silva, Michelle Álefe Alves Cury, Vinícius Toledo Couto, Raitza Araújo dos Santos Lima, Bárbara Sousa Modesto 538 Impact of cataracts and cataract surgery on quality of vision Eirini Skiadaresi, Colm McAlinden Cochrane highlights 540 Acupuncture for migraine prophylaxis Klaus Linde, Gianni Allais, Benno Brinkhaus, Eric Manheimer, Andrew Vickers, Adrian R. White Comments: Fabíola Dach 541 HMG CoA reductase inhibitors (statins) for people with chronic kidney disease not requiring dialysis Suetonia C. Palmer, Sankar D. Navaneethan, Jonathan C. Craig, David W. Johnson, Vlado Perkovic, Jorgen Hegbrant, Giovanni F. M. Strippoli Comments: Cássio José de Oliveira Rodrigues I III List of the names of the referees for the studies that were published in the São Paulo Medical Journal/Evidence for Health Care during the year 2015 Instructions for authors ( Sao Paulo Med J. 2015; 133(6):i-ii i

4 ORGANIZATION Founded in 1932, a bimonthly publication of the Associação Paulista de Medicina revistas@apm.org.br Editors: Paulo Andrade Lotufo and Álvaro Nagib Atallah. Editorial advisor: Rachel Riera. Editorial assistant: Marina de Britto. Scientific journalist and editor: Patrícia Logullo (MTB: ). Editorial auxiliary: Flávia Venezio. Associate editors: Adriana Seber, Aécio Flávio Teixeira de Góis, Airton Tetelbom Stein, Alexander Wagner Silva de Souza, Antonio José Gonçalves, Aytan Miranda Sipahi, Cristina Muccioli, Delcio Matos, Domingo Marcolino Braile, Edina Mariko Koga da Silva, Fernando Antonio de Almeida, Flávio Faloppa, Heráclito Barbosa de Carvalho, José Antônio Rocha Gontijo, José Carlos Costa Baptista-Silva, José Maria Soares Júnior, José Roberto Lapa e Silva, Laércio Joel Franco, Maria do Patrocínio Tenório Nunes, Milton de Arruda Martins, Moacir Fernandes de Godoy, Olavo Pires de Camargo, Renato Corrêa Baena, Sergio Tufik, Vania dos Santos Nunes. Proofreading: David Elliff. Desktop publishing: Zeppelini Editorial ( Listed in: Medline, Lilacs, SciELO, Science Citation Index Expanded and Journal Citation Reports/Sciences Edition (impact factor 0.588) and EBSCO publishing. International Board: Alexandre Wagner Silva de Souza (University Medical Center Groningen, Groningen, Netherlands), Charles J. Menkes (Cochin Hospital, Paris, France), José Fragata (Hospital Cuf Infant Santo, Lisbon), Luiz Dratcu (Guy s Hospital, London, and Maudsley NHS Trust, York Clinic, London), Marcelo Cypel (University Health Network, Toronto, Canada), Karla Soares-Weiser (Enhance Reviews Ltd, Wantage, United Kingdom), Tirone E. David (Toronto General Hospital, Toronto, Canada), Mário Viana de Queiroz (Hospital de Santa Maria, Lisbon), Wadih Arap (MD Anderson Cancer Center, University of Texas, Houston, United States), Wellington Cardoso (Boston University, Boston, United States). All articles published, including editorials and letters, represent the opinions of the authors and do not reflect the official policy of the Associação Paulista de Medicina or the institution with which the authors are affiliated, unless this is clearly specified. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Copyright 2015 by Associação Paulista de Medicina. SPMJ website: access to the entire São Paulo Medical Journal/Revista Paulista de Medicina website is free to all. We will give at least six months notice of any change in this policy. SPMJ printed version: six issues/year; 1 volume/year, beginning on first Thursday in January. One-year subscription for the year 2015: individual US$ 165; institutional US$ 230. Scientific Council Abrão Rapoport Hospital Heliópolis, São Paulo Adriana Costa e Forti Faculdade de Medicina, Universidade Federal do Ceará Alexandre Fogaça Cristante Faculdade de Medicina da Universidade de São Paulo Álvaro Nagib Atallah Escola Paulista de Medicina, Universidade Federal de São Paulo Auro del Giglio Faculdade de Medicina da Fundação ABC Carlos Alberto Morais Sá Universidade do Rio de Janeiro - UNIRIO Carmen Cabanelas Pazos de Moura Instituto Carlos Chagas Filho, Universidade Federal do Rio de Janeiro Cármino Antonio De Souza Faculdade de Ciências Médicas, Universidade Estadual de Campinas Dario Birolini Faculdade de Medicina, Universidade de São Paulo Eduardo Maia Freese de Carvalho Faculdade de Medicina, Universidade Federal de Pernambuco, Centro de Pesquisas Aggeu Magalhães - CpqAM/FIOCRUZ. Egberto Gaspar de Moura Instituto de Biologia Roberto Alcantara Gomes, Universidade Estadual do Rio de Janeiro Eliézer Silva Hospital Israelita Albert Einstein, São Paulo Emílio Antonio Francischetti - Faculdade de Medicina da Universidade Estadual do Rio de Janeiro Emmanuel de Almeida Burdmann Faculdade de Medicina de São José do Rio Preto Fabio Bessa Lima Instituto de Ciências Biomédicas, Universidade de São Paulo Florence Kerr-Corrêa Faculdade de Medicina de Botucatu, Universidade Estadual de São Paulo Francisco José Penna Faculdade de Medicina Universidade Federal de Minas Gerais Geraldo Rodrigues de Lima Escola Paulista de Medicina, Universidade Federal de São Paulo Irineu Tadeu Velasco Faculdade de Medicina da Universidade de São Paulo João Renato Rebello Pinho Instituto Adolfo Lutz, Secretaria de Estado da Saúde de São Paulo Joel Spadaro Faculdade de Ciências Médicas de Botucatu, Universidade Estadual de São Paulo Jorge Pinto Ribeiro Faculdade de Medicina, Universidade Federal do Rio Grande do Sul Jorge Sabbaga Hospital Alemão Oswaldo Cruz, São Paulo José Antonio Marin-Neto Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo José Carlos Nicolau Instituto do Coração, Universidade de São Paulo José Geraldo Mill Faculdade de Medicina, Universidade Federal do Espírito Santo José Mendes Aldrighi Faculdade de Saúde Pública, Universidade de São Paulo José Roberto Lapa e Silva Instituto de Doenças do Tórax, Universidade Federal do Rio de Janeiro Leonardo Roever Universidade Federal de Uberlândia Leopoldo Soares Piegas Instituto Dante Pazzanese de Cardiologia, São Paulo Luiz Jacintho da Silva Faculdade de Ciências Médicas, Universidade Estadual de Campinas Luiz Paulo Kowalski Hospital AC Camargo, São Paulo Márcio Abrahão Escola Paulista de Medicina, Universidade Federal de São Paulo Maria Inês Schmidt Faculdade de Medicina, Universidade Federal do Rio Grande do Sul Maurício Mota de Avelar Alchorne Escola Paulista de Medicina, Universidade Federal de São Paulo Mauro Schechter Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro Milton de Arruda Martins Faculdade de Medicina, Universidade de São Paulo Moysés Mincis Faculdade de Ciências Médicas de Santos Nelson Hamerschlak Hospital Israelita Albert Einstein, São Paulo Noedir Antônio Groppo Stolf Faculdade de Medicina, Universidade de São Paulo Pérsio Roxo Júnior Faculdade de Medicina de Ribeirão Preto Raul Cutait Hospital Sírio-Libanês, São Paulo Raul Negrão Fleury Instituto Lauro de Souza Lima, Coordenadoria dos Institutos de Pesquisa da Secretaria de Saúde de São Paulo Raul Marino Junior Faculdade de Medicina, Universidade de São Paulo Ricardo Brandt de Oliveira Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo Roberto A. Franken Faculdade de Ciências Médicas da Santa Casa de Misericórdia de São Paulo Ruy Laurenti Faculdade de Saúde Pública, Universidade de São Paulo Soubhi Kahhale Faculdade de Medicina, Universidade de São Paulo Wilson Roberto Catapani Faculdade de Medicina do ABC, Santo André Wilson Cossermelli Reclin Reumatologia Clínica, São Paulo Diretoria Executiva da Associação Paulista de Medicina (Triênio ) Presidente: Florisval Meinão 1 o Vice-Presidente: Roberto Lotfi Júnior 2 o Vice-Presidente: Donaldo Cerci da Cunha 3 o Vice-Presidente: Paulo de Conti 4 o Vice-Presidente: Akira Ishida Secretário Geral: Paulo Cezar Mariani 1 o Secretário: Antonio José Gonçalves Diretor Administrativo: Lacildes Rovella Júnior Diretor Administrativo Adjunto: Roberto de Mello 1 o Diretor de Patrimônio e Finanças: Carlos Alberto Martins Tosta 2 o Diretor de Patrimônio e Finanças: Claudio Alberto Galvão Bueno Da Silva Diretor Cientí fico: Paulo Andrade Lotufo Diretor Cientí fico Adjunto: Álvaro Nagib Atallah Diretor de Defesa Profi ssional: João Sobreira de Moura Neto Diretor de Defesa Pro fissional Adjunto: Marun David Cury Diretor de Comunicações: Ivan Melo de Araújo Diretor de Comunicações Adjunto: Amilcar Martins Giron Diretor de Marketing: Ademar Anzai Diretor de Marketing Adjunto: Nicolau D Amico Filho Diretora de Eventos: Mara Edwirges Rocha Gândara Diretora de Eventos Adjunta: Regina Maria Volpato Bedone Diretor de Tecnologia de Informação: Antônio Carlos Endrigo Diretor de Tecnologia de Informação Adjunto: Marcelo Ferraz de Campos Diretor de Previdência e Mutualismo: Paulo Tadeu Falanghe Diretor de Previdência e Mutualismo Adjunto: Clóvis Francisco Constantino Diretor Social: Alfredo de Freitas Santos Filho Diretora Social Adjunto: Christina Hajaj Gonzalez Diretora de Responsabilidade Social: Evangelina de Araujo Vormittag Diretor de Responsabilidade Social Adjunto: José Eduardo Paciência Rodrigues Diretor Cultural: Guido Arturo Palomba Diretor Cultural Adjunto: José Luiz Gomes do Amaral Diretora de Serviços aos Associados: Vera Lúcia Nocchi Cardim Diretor de Serviços aos Associados Adjunto: João Carlos Sanches Anéas Diretor de Economia Médica: Tomás Patrício Smith-Howard Diretora de Economia Médica Adjunta: Marly Lopes Alonso Mazzucato 1 o Diretor Distrital: Everaldo Porto Cunha 2 o Diretora Distrital: Lourdes Teixeira Henriques 3 o Diretor Distrital: Camillo Soubhia Júnior 4 o Diretor Distrital: Wilson Olegário Campagnone 5 o Diretor Distrital: Flavio Leite Aranha Junior 6 o Diretora Distrital: Cleusa Cascaes Dias 7 o Diretora Distrital: Irene Pinto Silva Masci 8 o Diretor Distrital: Helencar Ignácio 9 o Diretora Distrital: Margarete Assis Lemos 10 o Diretor Distrital: Enio Luiz Tenório Perrone 11 o Diretora Distrital: Zilda Maria Tosta Ribeiro 12 o Diretor Distrital: Luís Eduardo Andreossi 13 o Diretor Distrital: Marcio Aguilar Padovani 14 o Diretor Distrital: Marcelo Torrente Silva ii Sao Paulo Med J. 2015; 133(6):i-ii

5 DOI: / EDITORIAL Stroke is still a neglected disease in Brazil Acidente vascular cerebral permanece doença negligenciada no Brasil Paulo Andrade Lotufo I Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil I MD, DrPH. Full Professor, Department of Internal Medicine, Faculdade de Medicina da Universidade de São Paulo (FMUSP), São Paulo, Brazil. Data from the Brazilian Ministry of Health show that the absolute number of deaths due to coronary heart disease surpassed the number of some fatal cerebrovascular events in Brazil only in 2011, for all ages. Higher risk of death due to heart disease than due to cerebrovascular diseases is the pattern in the Western hemisphere. However, as pointed out ten years ago in this Journal, Brazil had the highest age-adjusted stroke death rate of all Latin-American countries. 1 Despite the decline in stroke death rate throughout this country, 2 the risk of premature death due to stroke in Brazil is one of the highest in the world. To prove this statement, we take data from the Global Burden of Disease 2013 study relating to mortality and years of life lost (YLL) due to premature death. 3,4 First, we compare the years of life lost due to coronary heart disease (CHD) and stroke in South America (except Guyana and Suriname). Second, we compare the YLL of Brazil with that of 18 other selected countries (total of 19 countries). For both comparisons, there is an average value and there are places that can be described as presenting three situations: significantly higher than the mean; indistinguishable from the mean and considerably lower than the mean. The YLL due to CHD is higher in Venezuela and Paraguay, and Brazil is ranked third, but with values indistinguishable from the mean (Figure 1a). However, the YLL for cerebrovascular diseases is considerably higher in Paraguay and Brazil (Figure 1b). (a) Coronary heart disease higher than the average average lower than the average Years of life lost Paraguay Venezuela Brazil Colombia Bolivia Average Argentina Ecuador Uruguay Peru Chile (b) Stroke higher than the average average lower than the average Years of life lost Paraguay Brazil Bolivia Average Uruguay Argentina Venezuela Colombia Ecuador Chile Peru Figure 1. Age-adjusted years of life lost (in thousands) for coronary heart disease (a) and stroke (b) among South American countries in Sao Paulo Med J. 2015; 133(6):

6 EDITORIAL Lotufo PA Comparison among the selected countries reveals that the YLL due to CHD in Brazil is situated at the mean of the other countries (Figure 2a). On the other hand, for YLL due to cerebrovascular diseases, Brazil is ranked seventh out of 19 countries, with values significantly higher than the mean (Figure 2b). The Global Burden of Disease study established an innovative analytical approach towards the official health statistics. 3,4 However, comparison of health statistics among countries has two main limitations. One is nosological, relating to the coverage of the mortality system and quality of the death certification, which can weaken these comparisons. The other relates to the fact that mortality information systems based on the underlying cause of death have a primary limitation with regard to competing causes of mortality between countries. For example, Bolivia and Ecuador have lower rates of CHD and stroke than Brazil. In contrast, the YLL due to respiratory infections and stomach cancer are significantly higher in Bolivia and Ecuador than in Brazil. Consequently, these Andean countries have lower probability of cardiovascular death than that of Brazil or other South American countries like Argentina. Apart from these important points, it is undeniable that the burden of stroke is still high in Brazil and that the cardiovascular epidemiological transition in Brazil has been delayed by the burden of stroke mortality. 5 One important factor in this regard relates to the social, racial and regional differences in stroke mortality in Brazil, in comparison with elsewhere, with high risk among the poorest segments, blacks and people living in northern and northeastern Brazil. 6-8 In relation to these two components of mortality, there is no conclusive data about their incidence in Brazil, but we can speculate that case fatality is declining because of the rise in the numbers of elderly people dying due to stroke sequelae. 2 Moreover, the Brazilian National Health Survey described point prevalences of self-reported stroke of 1.6% and 1.4%, among men and women, respectively. The prevalences of post-stroke disabilities were 29.5% for men and 21.5% for women. 9 (a) Coronary heart disease higher than the average average lower than the average Years of life lost Russia India Indonesia Saudi Arabia Turkey Brazil Average United States Argentina China Mexico South Africa Germany Canada United Kingdom Australia Italy France South Korea Japan (b) Stroke higher than the average average lower than the average Years of life lost Indonesia Russia China India South Africa Brazil Saudi Arabia Average Turkey Argentina South Korea Mexico Japan United Kingdom Germany Italy United States Australia France Canada Figure 2. Age-adjusted years of life lost (in thousands) for coronary heart disease (a) and stroke (b) among 19 selected countries in Sao Paulo Med J. 2015; 133(6):457-9

7 Stroke is still a neglected disease in Brazil EDITORIAL Recently, an editorial in the journal Arquivos de Neuro- Psiquiatria signed by Fernandes provided a very good summary of the situation of policies for halting the stroke burden in Brazil. Fernandes rightly stated that we are doing badly due to delays and an inability to implement what is known (cost-effective prevention), causing suffering (morbidity), loss of many lives (mortality) and financial loss. Furthermore, he concluded that there has been insufficient investment in evaluating the effects of populational and non-pharmacological interventions, health services for people with CVD are poorly organized. 10 The first editorial about stroke as a neglected disease in Brazil was published in We are hoping to be invited in 2025 to write a narrative with a tentative title of How Brazil did the right thing towards curbing stroke mortality. Date of first submission: October 5, 2015 Last received: October 5, 2015 Accepted: October 5, 2015 Address for correspondence: Paulo Andrade Lotufo Centro de Pesquisa Clínica e Epidemiológica, Hospital Universitário, Universidade de São Paulo Av. Prof. Lineu Prestes, Butantã São Paulo (SP) Brasil Tel. (+55 11) palotufo@hu.usp.br REFERENCES 1. Lotufo PA. Stroke in Brazil: a neglected disease. Sao Paulo Med J. 2005;123(1): Lotufo PA, Goulart AC, Fernandes TG, Benseñor IM. A reappraisal of stroke mortality trends in Brazil ( ). Int J Stroke. 2013;8(3): GBD 2013 Mortality and Causes of Death Collaborators. Global, regional, and national age-sex specific all-cause and cause-specific mortality for 240 causes of death, : a systematic analysis for the Global Burden of Disease Study Lancet. 2015;385(9963): GBD 2013 DALYs and HALE Collaborators, Murray CJ, Barber RM. Global, regional, and national disability-adjusted life years (DALYs) for 306 diseases and injuries and healthy life expectancy (HALE) for 188 countries, : quantifying the epidemiological transition. Lancet. 2015;pii:S (15):61340-X. 5. Lotufo PA, Benseñor IM. Stroke mortality in Brazil: one example of delayed epidemiological cardiovascular transition. Int J Stroke (1): Vincens N, Stafström M. Income Inequality, Economic Growth and Stroke Mortality in Brazil: Longitudinal and Regional Analysis PLoS One. 2015;10(9):e Fernandes TG, Bando DH, Alencar AP, Benseñor IM, Lotufo PA. Income inequalities and stroke mortality trends in Sao Paulo, Brazil, Int J Stroke Jun 4. doi: /ijs [Epub ahead of print]. 8. Lotufo PA, Bensenor IJ. Raça e mortalidade cerebrovascular no Brasil [Race and stroke mortality in Brazil]. Rev Saude Publica. 2013;47(6): Bensenor IM, Goulart AC, Szwarcwald CL, et al. Prevalence of stroke and associated disability in Brazil: National Health Survey Arq Neuropsiquiatr. 2015;73(9): Fernandes JG. Stroke prevention and control in Brazil: missed opportunities. Arq Neuropsiquiatr. 2015;73(9): Sources of funding: Not declared Conflict of interest: Honoraria from Abbvie Brazil and Abbott Brazil for lectures. Sao Paulo Med J. 2015; 133(6):

8 ORIGINAL ARTICLE DOI: / Assessment of a glycated hemoglobin point-of-care analyzer (A1CNow+) in comparison with an immunoturbidimetric method: a diagnostic accuracy study Avaliação de um aparelho de diagnóstico local para hemoglobina glicada (A1CNow+) comparado com um método imunoturbidimétrico: estudo de acurácia de teste diagnóstico Aurélie Affret I, Luiz Henrique Maciel Griz II, Eduarda Ângela Pessoa Cesse III, Yuri da Silva Specht IV, Eduardo Maia Freese de Carvalho V, Annick Fontbonne VI Aggeu Magalhães Research Centre, Fundação Oswaldo Cruz (Fiocruz), and data were collected at the Department of Endocrinology, Agamemnon Magalhães Hospital, Universidade de Pernambuco (UPE), Recife, Pernambuco, Brazil I BSc. Master s Student in Nutrition, Department of Nutrition and Associated Pathologies, Institut de Recherche pour le Développement (IRD), Universités Montpellier, Montpellier, France. II MD, PhD. Professor, Department of Endocrinology, Agamemnon Magalhães Hospital, Universidade de Pernambuco, Pernambuco, Recife, Brazil. III PhD. Epidemiology and Public Health Researcher, Community Health Department, Aggeu Magalhães Research Center, Fundação Oswaldo Cruz (Fiocruz), Pernambuco, Recife, Brazil. IV Undergraduate in Statistics, Community Health Department, Aggeu Magalhães Research Center, Fundação Oswaldo Cruz (Fiocruz), Pernambuco, Recife, Brazil. V MD, PhD. Epidemiology and Public Health Researcher, Community Health Department, Aggeu Magalhães Research Center, Fundação Oswaldo Cruz (Fiocruz), Pernambuco, Recife, Brazil. VI MD, PhD. Epidemiology and Public Health Researcher, Department of Nutrition and Associated Pathologies, Institut de Recherche pour le Développement (IRD), Universités Montpellier, Montpellier, France. KEY WORDS: Hemoglobin A, glycosylated. Point-of-care systems. Technology assessment, biomedical. Diabetes mellitus. Primary health care. PALAVRAS-CHAVE: Hemoglobina A glicosilada. Sistemas automatizados de assistência junto ao leito. Avaliação da tecnologia biomédica. Diabetes mellitus. Atenção primária à saúde. ABSTRACT CONTEXT AND OBJECTIVE: To monitor glycemic control in diabetic patients, regular measurement of glycated hemoglobin (HbA1c) is recommended, but this can be difficult in remote places without access to laboratories. Portable point-of-care testing devices can prove a useful alternative. Our study aimed to assess the performance of one of them: A1CNow+, from Bayer. DESIGN AND SETTING: Cross-sectional accuracy study conducted at a university hospital in Brazil. METHODS: We made three successive measurements of capillary HbA1c using the A1CNow+ in 55 diabetic volunteers, while the same measurement was made on venous blood using the hospital reference method (Vitros 5,1 FS). We used the Bland-Altman graphical method to assess the A1CNow+ in relation to the Vitros 5,1 FS method. We also evaluated clinical usefulness by calculating the sensitivity and specificity of A1CNow+ for detecting patients with HbA1c lower than 7%, which is the usual limit for good glycemic control. RESULTS: The coefficient of variation between repeat testing for the A1CNow+ was 3.6%. The mean difference between A1CNow+ and Vitros 5,1 FS was +0.67% (95% confidence interval, CI: to +0.81). The agreement limits of our Bland-Altman graph were (95% CI: to -0.19) and (95% CI: to +2.05). The sensitivity and specificity in relation to the 7% limit were respectively 100% and 67.7%. CONCLUSIONS: Although the A1CNow+ had good sensitivity, its accuracy was insufficient for use as a replacement for laboratory measurements of HbA1c, for glycemic control monitoring in diabetic patients. RESUMO CONTEXTO E OBJETIVO: Para monitorar o controle glicêmico dos diabéticos, é recomendado medir regularmente a hemoglobina glicada (HbA1c). Isso pode ser difícil em locais distantes sem acesso a laboratórios. Uma alternativa é usar aparelhos portáteis à beira do leito do paciente. Nosso estudo visou avaliar o desempenho de um deles: A1CNow+, da Bayer. TIPO DE ESTUDO E LOCAL: Estudo transversal de acurácia realizado em hospital universitário no Brasil. MÉTODOS: Medimos, com o A1CNow+, três vezes seguidas, a HbA1c capilar de 55 diabéticos voluntários, enquanto a mesma medida era feita em sangue venoso pelo método de referência do hospital (Vitros 5,1 FS). Usamos a análise gráfica de Bland-Altman para avaliar o A1CNow+ em relação ao Vitros 5,1 FS. Verificamos a utilidade clínica através do cálculo da sensibilidade e da especificidade do A1CNow+ para detectar pacientes com HbA1c abaixo de 7%, limite usual indicando glicemia controlada. RESULTADOS: O coeficiente de variação entre testes repetidos do A1CNow+ foi de 3,6%. A diferença média entre o A1CNow+ e o Vitros 5,1 FS foi de +0,67% (95% intervalo de confiança, IC: +0,52 para +0,81). Os limites de concordância do gráfico de Bland-Altman foram -0,45 (95% IC: -0,71 para -0,19) and +1,82 (95% IC: +1,52 para +2,05). A sensibilidade e a especificidade em relação ao limite de 7% foram 100% e 67,7%, respectivamente. CONCLUSÃO: Apesar da boa sensibilidade, o A1CNow+ não tem acurácia suficiente para ser utilizado no monitoramento do controle glicêmico de pacientes diabéticos em substituição das medidas da HbA1c em laboratório. 460 Sao Paulo Med J. 2015; 133(6):460-4

9 Assessment of a glycated hemoglobin point-of-care analyzer (A1CNow+) in comparison with an immunoturbidimetric method a diagnostic accuracy study ORIGINAL ARTICLE INTRODUCTION Diabetes mellitus is a major public health issue all over the world. In the International Diabetes Federation (IDF) estimates for 2012, 1 Brazil has a diabetes prevalence of 10.52%, which is higher than in the United States, where the prevalence is 9.35%. The prevalence of self-reported diabetes in the populations of Brazilian state capitals over 18 years of age was 6.3% in From 1950 to 2007, mortality trends due to diabetes increased in most Brazilian state capitals. 3,4 Part of this observed increase can be attributed to improvements in access to diagnosis and death certification, but it stresses the importance of improving diabetes prevention and control. 4 To fight diabetes and other chronic diseases, Brazil is developing strategies within its National Health System (Sistema Único de Saúde, SUS), one of the largest public health systems in the world. 5 Within SUS, the Family Health Strategy (Estratégia Saúde da Família, ESF) is in charge of most of Brazilian primary care. In 2001, the Brazilian Ministry of Health launched the Plan for the Reorganization of Care for Arterial Hypertension and Diabetes Mellitus. 6 Among the interventions that it supported was regular determination of glycated hemoglobin (HbA1c) levels at ESF primary care units among patients with diabetes. HbA1c provides clinicians with an indication of a patient s average blood glucose level over the past two to three months and measurement of this parameter is recommended for assessment of diabetes control. HbA1c also helps estimate the risk of developing diabetes-associated micro and macrovascular complications. This was shown by the results obtained in the Diabetes Control and Complication Trial (DCCT) and United Kingdom Prospective Diabetes Study (UKPDS). 7,8 Following these results, the American Diabetes Association (ADA) and other national bodies issuing guidelines for diabetes treatment recommended that one primary goal of therapy should be to maintain HbA1c below 7%. 9 The ADA also recommended that HbA1c testing should be performed at least biannually in all patients and quarterly for patients whose therapy has changed or who are not meeting treatment goals. 9 Brazilian recommendations advise one measurement per patient every three months. 6 However, in many regions of Brazil, these recommendations are difficult to achieve because of a lack of medical analysis laboratories able to meet patients needs, especially in areas distant from urban centers. This is the case in the state of Pernambuco, in the northeastern region of Brazil, even though the ESF is well established there. Moreover, this situation is not specific to Brazil but can be found in many developing countries where there are remote places with poor access to infrastructure and healthcare professionals. Therefore, HbA1c measurement by means of point-of-care testing (PoCT), directly performed by primary care unit staff, could be a useful alternative to measurement at a medical analysis laboratory. OBJECTIVE The aim of this study was to assess the performance of A1CNow+, a PoCT device from Bayer (São Paulo, Brazil), in comparison with the Vitros 5,1 FS method, an immunoturbidimetric laboratory method used in a university hospital in Recife, Pernambuco. Specifically, the objective was to determine whether it could constitute a useful alternative to laboratory measurement, in order to monitor diabetes control in primary care units in remote areas. Our hypothesis was that values of HbA1c given by the PoCT device would be close enough to values given by the immunoturbidimetric method to allow correct decision-making for blood glucose control, in terms of possible modifications of antidiabetic treatments (pharmacological or non-pharmacological). METHODS A cross-sectional accuracy study was conducted between June and July 2012 at the endocrinology department of one of the state university hospitals, in Recife, state of Pernambuco, northeastern region of Brazil. Outpatients or inpatients at the department were consecutively approached to be invited to participate, if they were 18 years of age or older, had known diabetes, had a prescription to undergo a venous blood test at the hospital with HbA1c measurement, were not pregnant (for women) and did not have any known hemoglobinopathy, anemia and/or end-stage renal failure. Fifty-five patients accepted the invitation to participate. All of them provided informed consent and completed a short questionnaire to gather their baseline characteristics. Then, a venous blood test was performed by a hospital nurse and sent to the hospital laboratory for HbA1c to be measured using the Vitros 5,1 FS method, which was the current HbA1c measurement method used at the hospital and therefore used as the reference against which we evaluated the A1CNow+. The Vitros 5,1 FS method uses venous blood samples and is standardized against the International Federation of Clinical Chemistry (IFCC) reference system for HbA1c and aligned to the DCCT standards through the National Glycohemoglobin Standardization Program (NGSP). 10 At the same time, their capillary HbA1c was also measured three consecutive times using the A1CNow+, by a master s student who had been trained to perform the method and who had no knowledge of the Vitros 5,1 FS result, since the latter was only returned a few days later. The A1CNow+ is also standardized against the IFCC and aligned to the DCCT standards via the NGSP. It uses both immunoassay and chemistry technology to measure HbA1c and total hemoglobin, respectively, and provides results in five minutes. It is small (length * width * height in mm: 53 * 80 * 15), light and easy to use; it does not require calibration and can be stored at room temperatures up to 50 C (an important asset for tropical climates, such as in Pernambuco). It is approved for commercialization in Brazil. Sao Paulo Med J. 2015; 133(6):

10 ORIGINAL ARTICLE Affret A, Griz LHM, Cesse EAP, Specht YS, Carvalho EMF, Fontbonne A To assess the reproducibility of A1CNow+ measurements, their coefficient of variation (defined as the ratio of the standard deviation to the mean) was calculated using the measurements performed on each patient. For this calculation, only results from patients from whom we obtained at least two valid HbA1c measurements were taken into consideration. We did not use weighted means because of their lack of impact in this study. Bland and Altman analysis was used to assess agreement between the two methods. 11 According to these authors, this analysis requires a sample size of at least 50 units, which determined our choice of number of patients. The differences between the methods (A1CNow+ minus Vitros 5,1 FS) were plotted against the average of the two measurements. The size of the differences and their distribution around zero were tested. Finally, to assess the clinical usefulness of A1CNow+, its sensitivity and specificity were calculated in relation to the threshold of 7%, which is the usually recommended limit for diabetes control. 9 For both the agreement and the usefulness analyses, only the first HbA1c measurement obtained from each patient using the A1CNow+ was used in the calculation, since this is what would happen if the PoCT was used under routine clinical conditions. Statistical analyses were performed using Epi Info and the Statistical Package for the Social Sciences (SPSS), version 19 (SPSS Inc., Chicago, IL, USA). The study was approved by the Ethics Committee of the Aggeu Magalhães Research Center, Oswaldo Cruz Foundation (CPqAM/Fiocruz) and the Brazilian National Commission for Research Ethics (CONEP). All participating subjects were duly informed of the objectives and procedures of the study, and all of them signed an informed consent form. RESULTS Most of the patients were women (50 out of 55). Their mean age was 61.3 ± 13.0 years (range: 20-85). Their mean body mass index (BMI) was 27.8 ± 4.2 kg/m 2, and 75% of the patients were overweight (25 BMI < 30 kg/m 2 ) or obese (BMI 30 kg/m 2 ). The mean duration of diabetes was 10.1 ± 8.6 years; 85.5% of the patients were using oral antidiabetic agents and 30.9% were taking insulin injections, alone or combined with oral antidiabetic therapy. From the laboratory method, the mean for HbA1c measurements was 7.26 ± 1.87%. From A1CNow+, the mean for HbA1c measurements was 7.93 ± 1.97%, just considering the first measurement, and 7.91 ± 1.89% considering all measurements. The coefficient of variation for repeated measurements on the same patient was 3.6%. Bland-Altman analysis of accuracy showed that the mean difference between the A1CNow+ and the Vitros 5,1 FS determinations was +0.67% (95% confidence interval, CI: +0.52% to +0.81%) and the standard deviation of the differences was 0.56%, such that A1CNow+ consistently gave higher measurements than the laboratory method (Figure 1). The lower and upper limits of agreement of the graph were -0.45% (95% CI: -0.71% to -0.19%) and (95% CI: to +2.05). The sensitivity was 100% and specificity was 67.7% in relation to the 7% level (Table 1). It appeared that 18.2% of the patients (10 patients) were incorrectly classified using the analyzer. All of them were false positive : they had HbA1c 7% with A1CNow+, whereas their HbA1c was < 7% with the Vitros 5,1 FS method. DISCUSSION PoCT has been advocated with the objective, among others, of promoting faster professional decisions and facilitating patients adherence to medical counseling. 12 Indeed, in case of diabetes, studies have shown the benefits of rapid HbA1c results at the time of the patient s visit by improving glucose control through intensification of therapy, 13,14 although controversy currently exists regarding this evidence and the analytical quality relative to laboratory testing. 15 Another potential advantage of PoCT is as a replacement for laboratory measurements in contexts in which laboratory facilities are scarce and often concentrated in urban areas, thus limiting access to populations and jeopardizing quality of care. 16,17 This is the case in many developing countries, and Brazil, with its continental dimensions, is no exception. Difference (HbA1c%) = (A1CNow+ - Vitros 5,1 FS) Mean (HbA1c%) = (A1CNow+ + Vitros 5,1 FS)/2 Figure 1. Bland-Altman difference plot comparing the A1CNow+ versus the Vitros 5,1 FS method. Table 1. Patients distribution according to their HbA1c results from the A1CNow+ and Vitros 5,1 FS methods, in relation to the 7% level Laboratory method (Vitros 5,1 FS) HbA1c 7% HbA1c < 7% HbA1c 7% A1CNow+ HbA1c < 7% Sao Paulo Med J. 2015; 133(6):460-4

11 Assessment of a glycated hemoglobin point-of-care analyzer (A1CNow+) in comparison with an immunoturbidimetric method a diagnostic accuracy study ORIGINAL ARTICLE However, PoCT devices need to be proven to provide sufficiently reliable measurements in order to be recommended for use in cases of lack of or difficulty in access to laboratory services. 18 The present study investigated the analytical performance of the PoCT A1CNow+, a device that has been approved for use in Brazil, comparing it with the Vitros 5,1 FS laboratory method. The latter acted as our reference method, since it is the method used in the state university hospital where we conducted this evaluation, which is the reference hospital for diabetes in the state of Pernambuco. The coefficient of variation of the A1CNow+ device was found to be 3.6%. This result was in agreement with some previous reports, 19 and lower than other results. 20 Although it has been recommended that HbA1c assays should have a coefficient of variation of < 2%, 21 this criterion is very strict and difficult to meet, even for certain laboratory-based methods. 22 It would therefore seem inappropriate to impose this goal on PoCT devices measuring HbA1c. Earlier reports recommended that HbA1c assays should have a coefficient of variation of < 5% (ideally < 3%), 23 in which case our study revealed the A1CNow+ device to be satisfactory in terms of reproducibility. However, in terms of accuracy, the Bland-Altman analysis did not demonstrate good agreement between the analyzer and the laboratory method: the results from the A1CNow+ device were systematically higher. The limits of agreement of the graph were -0.45% to +1.79%, thus not fulfilling one of the required NGSP criteria, which is that the 95% CI of the difference between the tested method and the NGSP be ± 0.75% of HbA1c. 10 Although one study reported an even greater discrepancy between A1CNow+ results and the reference method, 20 others have shown better agreement, with mean differences of between -0.20% and -0.10%, comparing A1CNow+ with the internationally accepted reference laboratory method of highperformance liquid chromatography (HPLC). 19 In fact, the discrepancy that we found may possibly have been due to the Vitros 5,1 FS method, about which little information is available in the literature. This is indeed the most important limitation of this study: the A1CNow+ results were compared with those obtained from the Vitros 5,1 FS laboratory method and not the internationally accepted HPLC reference laboratory method, which was used in the DCCT and the UKPDS studies. 7,8 Nonetheless, it is important to note that we compared A1CNow+ with the laboratory method that was used in the reference hospital for diabetes in Pernambuco, which ensures highly specialized care for diabetic patients from all over the state. Therefore, there is reason to suppose that the hospital would use a high-standard laboratory method for assessing one of the most important parameters for monitoring blood glucose control. In keeping with the higher readings in relation to the laboratory method, the A1CNow+ device had good sensitivity (100%). However, its specificity was low (67.7%) in relation to the HbA1c level of 7%. This means that under clinical conditions, use of the A1CNow+ cannot be recommended: the false-positive rate was 10/55 (18.2%), and these patients consequently would have a risk of hypoglycemia if their treatment was intensified using the results from this analyzer. These results were quite similar to those obtained by Arrendale et al., 24 in which the sensitivity was 95% and the specificity was 74%. Accordingly, in addition to the desirability of comparing our results with those of the HPLC method, it might also be interesting in further research to investigate whether A1CNow+ always gives higher results than any laboratory method, in a constant manner. If this were the case, as Arrendale et al. suggested in their study, 24 it would still be possible to use this analyzer by applying a correction to the data obtained from it, which would consist of a calculation of the following form: expected laboratory HbA1c = a + b [HbA1c from A1CNow+]. This would be important for diabetic patients living in remote areas with poor access to infrastructure and healthcare professionals, which is often the case in Brazil. These patients would benefit from adequate glycemic control monitoring (HbA1c measurement two to four times a year) when access to venous blood testing in laboratories is difficult. For such purposes, devices like A1CNow+ could become important tools in primary care services. CONCLUSION According to the results from our study, A1CNow+ cannot be recommended for replacing laboratory measurements of HbA1c for glycemic control monitoring among diabetic patients, because the accuracy and specificity of its measurements were insufficient, compared with the method used in a reference university hospital. Further research is needed in order to compare its results with those obtained from a HPLC reference laboratory method, and/or to assess whether it could be used with the aid of a correction equation. REFERENCES 1. International Diabetes Federation. IDF Diabetes Atlas. 5 th ed., 2012 Update. Available from: IDFAtlasPoster_2012_EN.pdf. Accessed in 2014 (Sep 10). 2. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde. Secretaria de Gestão Estratégica e Participativa. Vigitel Brasil 2010: vigilância de fatores de risco e proteção para doenças crônicas por inquérito telefônico/ministério da Saúde, Secretaria de Vigilância em Saúde, Secretaria de Gestão Estratégica e Participativa. Brasilia: Ministério da Saúde; Available from: wordpress.com/2014/05/vigitel_2010.pdf. Accessed in 2014 (Sep 10). Sao Paulo Med J. 2015; 133(6):

12 ORIGINAL ARTICLE Affret A, Griz LHM, Cesse EAP, Specht YS, Carvalho EMF, Fontbonne A 3. Cesse EA, Carvalho EF, Souza WV, Luna CF. Tendência da mortalidade por diabetes melito no Brasil: 1950 a 2000 [Mortality trends by the diabetes mellitus in Brazil: 1950 to 2000]. Arq Bras Endocrinol Metabol. 2009;53(6): Mattos PE, Luz LL, Santiago LM, Mattos IE. Tendência da mortalidade por diabetes melito em capitais brasileiras, [Trends in mortality of diabetes mellitus patients in Brazilian capitals, ]. Arq Bras Endocrinol Metabol. 2012;56(1): Brasil. Portal da Saúde SUS. Cidadão, entenda o SUS. Available from: Accessed in 2014 (Dec 16). 6. Brasil. Ministério da Saúde. Secretaria de Políticas de Saúde. Departamento de Ações Programáticas Estratégicas. Plano de reorganização da atenção à hipertensão arterial e ao diabetes mellitus: hipertensão arterial e diabetes mellitus/departamento de Ações Programáticas Estratégicas. Brasilia: Ministério da Saúde; Available from: miolo2002.pdf. Accessed in 2014 (Sep 10). 7. The effect of intensive treatment of diabetes on the development and progression of long-term complications in insulin-dependent diabetes mellitus. The Diabetes Control and Complications Trial Research Group. N Engl J Med. 1993;329(14): Intensive blood-glucose control with sulphonylureas or insulin compared with conventional treatment and risk of complications in patients with type 2 diabetes (UKPDS 33). UK Prospective Diabetes Study (UKPDS) Group. Lancet. 1998;352(9131): American Diabetes Association. Standards of medical care in diabetes Diabetes Care. 2010;33 Suppl 1:S National Glycohemoglobin Standardization Program. Harmonizing Hemoglobin A1c Testing. More About HbA1c. Clinical use. Available from: Accessed in 2014 (Sep 11). 11. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet. 1986;1(8476): Price CP. Point of care testing. BMJ. 2001;322(7297): Cagliero E, Levina EV, Nathan DM. Immediate feedback of HbA1c levels improves glycemic control in type 1 and insulin-treated type 2 diabetic patients. Diabetes Care. 1999;22(11): Miller CD, Barnes CS, Phillips LS, et al. Rapid A1c availability improves clinical decision-making in an urban primary care clinic. Diabetes Care. 2003;26(4): Gialamas A, St John A, Laurence CO, Bubner TK; PoCT Management Committee. Point-of-care testing for patients with diabetes, hyperlipidaemia or coagulation disorders in the general practice setting: a systematic review. Fam Pract. 2010;27(1): Yager P, Domingo GJ, Gerdes J. Point-of-care diagnostics for global health. Annu Rev Biomed Eng. 2008;10: Martin DD, Shephard MD, Freeman H, et al. Point-of-care testing of HbA1c and blood glucose in a remote Aboriginal Australian community. Med J Aust. 2005;182(10): Martin CL. Quality control issues in point of care testing. Clin Biochem Rev. 2008;29 Suppl 1:S Bode BW, Irvin BR, Pierce JA, Allen M, Clark AL. Advances in hemoglobin A1c point of care technology. J Diabetes Sci Technol. 2007;1(3): Little RR, Lenters-Westra E, Rohlfing CL, Slingerland R. Point-of-care assays for hemoglobin A(1c): is performance adequate? Clin Chem. 2011;57(9): Sacks DB, Arnold M, Bakris GL, et al. Executive summary: guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clin Chem. 2011;57(6): Lenters-Westra E, Slingerland RJ. Six of eight hemoglobin A1c pointof-care instruments do not meet the general accepted analytical performance criteria. Clin Chem. 2010;56(1): Sacks DB, Bruns DE, Goldstein DE, et al. Guidelines and recommendations for laboratory analysis in the diagnosis and management of diabetes mellitus. Clin Chem. 2002;48(3): Arrendale JR, Cherian SE, Zineh I, Chirico MJ, Taylor JR. Assessment of glycated hemoglobin using A1CNow+ point-of-care device as compared to central laboratory testing. J Diabetes Sci Technol. 2008;2(5): Sources of funding: Conselho Nacional de Desenvolvimento Científico e Tecnológico CNPq, grant # / Conflict of interest: None Date of first submission: June 13, 2014 Last received: August 7, 2014 Accepted: September 11, 2014 Address for correspondence: Annick Fontbonne IRD, UMR 204 Nutripass 911 avenue d Agropolis B.P Cedex Montpellier - 95, France annick.fontbonne@ird.fr 464 Sao Paulo Med J. 2015; 133(6):460-4

13 DOI: / ORIGINAL ARTICLE Bacterial vaginosis in pregnant adolescents: proinflammatory cytokine and bacterial sialidase profile. Cross-sectional study Vaginose bacteriana em gestantes adolescentes: perfil de citocinas proinflamatórias e sialidases bacterianas. Estudo transversal Carolina Sanitá Tafner Ferreira I, Camila Marconi II, Cristina Maria de Lima Garcia Parada III, Marli Teresinha Cassamassimo Duarte IV, Ana Paula Oliveira Gonçalves V, Marilza Vieira Cunha Rudge VI, Márcia Guimarães da Silva VII Department of Nursing, Universidade Federal do Pará (UFPA), Belém, Pará, and Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (Unesp), Botucatu, São Paulo, Brazil I BSc, Master s Student, Department of Pathology, Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (Unesp), Botucatu, São Paulo, Brazil. II BSc, MSc, PhD. Postdoctoral Fellow. Department of Pathology, Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (Unesp), Botucatu, São Paulo, Brazil. III BSN, MSc, PhD. Adjunct Professor, Department of Nursing, Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (Unesp), Botucatu, São Paulo, Brazil. IV BSN, MSc, PhD. Assistant Professor, Department of Nursing, Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (Unesp), Botucatu, São Paulo, Brazil V BSN, MSc. Assistant Professor, Department of Nursing, Universidade Federal do Pará (UFPA), Belém, Pará, Brazil. VI MD, MSc, PhD. Titular Professor, Department of Gynecology and Obstetrics, Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (Unesp), Botucatu, São Paulo, Brazil. VII BSc, MSc, PhD. Assistant Professor, Department of Pathology, Faculdade de Medicina de Botucatu (FMB), Universidade Estadual Paulista (Unesp), Botucatu, São Paulo, Brazil. KEY WORDS: Pregnancy. Adolescent. Vaginosis, bacterial. Cytokines. Neuraminidase. PALAVRAS-CHAVE: Gravidez. Adolescente. Vaginose bacteriana. Citocinas. Neuraminidase. ABSTRACT CONTEXT AND OBJECTIVE: Bacterial vaginosis occurs frequently in pregnancy and increases susceptibility to sexually transmitted infections (STI). Considering that adolescents are disproportionally affected by STI, the aim of this study was to evaluate the cervicovaginal levels of interleukin (IL)-1 beta, IL-6, IL-8 and bacterial sialidase in pregnant adolescents with bacterial vaginosis. DESIGN AND SETTING: Cross-sectional study at mother and child referral units in Belém, Pará, Brazil. METHODS: Vaginal samples from 168 pregnant adolescents enrolled were tested for trichomoniasis and candidiasis. Their vaginal microbiota was classified according to the Nugent criteria (1991) as normal, intermediate or bacterial vaginosis. Cervical infection due to Chlamydia trachomatis and Neisseria gonorrhoeae was also assessed. Cytokine and sialidase levels were measured, respectively, using enzyme-linked immunosorbent assays and MUAN conversion in cervicovaginal lavages. Forty-eight adolescents (28.6%) were excluded because they tested positive for some of the infections investigated. The remaining 120 adolescents were grouped according to vaginal flora type: normal (n = 68) or bacterial vaginosis (n = 52). Their cytokine and sialidase levels were compared between the groups using the Mann-Whitney test (P < 0.05). RESULTS: The pregnant adolescents with bacterial vaginosis had higher levels of IL-1 beta, IL-6 and IL-8 (P < 0.05). Sialidase was solely detected in 35 adolescents (67.2%) with bacterial vaginosis. CONCLUSIONS: Not only IL-1 beta and sialidase levels, but also IL-6 and IL-8 levels are higher in pregnant adolescents with bacterial vaginosis, thus indicating that this condition elicits a more pronounced inflammatory response in this population, which potentially increases vulnerability to STI acquisition. RESUMO CONTEXTO E OBJETIVO: A vaginose bacteriana é uma condição, comum em gestantes, que aumenta a susceptibilidade a infecções sexualmente transmissíveis (IST). Considerando que adolescentes são desproporcionalmente afetadas por IST, o objetivo deste estudo foi avaliar os níveis cervicovaginais de interleucina (IL)-1 beta, IL-6, IL-8 e sialidases bacterianas em gestantes adolescentes com vaginose bacteriana. DESENHO DO ESTUDO E LOCAL: Estudo transversal em Unidade de Referência Materno Infantil (UREMIA), Belém, Pará, Brasil. MÉTODOS: Amostras vaginais das 168 gestantes adolescentes incluídas foram testadas para tricomoníase e candidíase e a microbiota vaginal foi classificada em normal, intermediária e vaginose bacteriana, segundo os critérios de Nugent (1991). Infecções cervicais por Chlamydia trachomatis e Neisseria gonorrhoeae também foram avaliadas. Os níveis de citocinas e sialidades foram quantificados, respectivamente, por método imunoenzimático e pela conversão do MUAN nos lavados cervicovaginais. Foram excluídas 48 (28,6%) adolescentes positivas para alguma das infecções investigadas. As 120 gestantes remanescentes foram agrupadas de acordo com o padrão de flora vaginal em: normal (n = 68) e vaginose bacteriana (n = 52). Níveis de citocinas e sialidases foram comparados pelo teste de Mann-Whitney, P < 0,05. RESULTADOS: As gestantes adolescentes com vaginose bacteriana entre os grupos apresentaram níveis aumentados de IL-1 beta, IL-6 and IL-8 (P < 0,05). Sialidases foram exclusivamente detectadas em 35 (67,2%) adolescentes com vaginose bacteriana. CONCLUSÕES: Não apenas a IL-1 beta e as sialidases estão aumentadas em gestantes adolescentes com vaginose bacteriana, mas também IL-6 e IL-8, indicando resposta inflamatória mais pronunciada dessa alteração de microbiota nesta população, potencializando a vulnerabilidade à aquisição de IST. Sao Paulo Med J. 2015; 133(6):

14 ORIGINAL ARTICLE Ferreira CST, Marconi C, Parada CMLG, Cassamassimo Duarte MT, Gonçalves APO, Rudge MVC, Silva MG INTRODUCTION Bacterial vaginosis is the most common type of abnormal vaginal flora and it is frequent in both pregnancy and adolescence. 1-3 In bacterial vaginosis, vaginal lactobacilli are replaced by other bacterial species, mostly anaerobes. 4 It has been consistently shown in the literature that bacterial vaginosis is strongly associated with increased risk of preterm delivery and acquisition of sexually transmitted infections (STIs). 1,5-7 The mechanisms underlying the associations between bacterial vaginosis and poor pregnancy outcomes and higher vulnerability to STIs remain to be addressed, but it is well established that alterations to vaginal immunity play a crucial role in them. 8,9 In fact, pregnancy and adolescence are both conditions associated with changes to the levels of immune mediators in the lower genital tract Typically, bacterial vaginosis is associated with increased cervicovaginal levels of interleukin (IL)-1 beta, but in pregnant women the levels of IL-6 and IL-8 are also elevated in the presence of bacterial vaginosis There is still a lack of information in the literature regarding the immune response to bacterial vaginosis in adolescents. However, it has already been demonstrated that the vaginal fluids of adolescents present altered levels of several immune mediators and show lower antimicrobial activity in vitro, in comparison with adults. 12 These findings are particularly important given that this population is disproportionally more affected by STIs. 17 Higher vaginal sialidase levels are another factor associated with increased vulnerability to STIs during bacterial vaginosis. Bacterial sialidase is capable of degrading local immunoglobulins and consequently compromising the local immune defense. 18 Additionally, elevated vaginal sialidase levels in early pregnancy has already been correlated with elevated risk of occurrence of spontaneous preterm labor. 19 Considering the importance of balanced vaginal immunity for protecting the lower genital tract against STIs, better understanding of the local response to bacterial vaginosis in pregnant adolescents may contribute towards new strategies for preventing infection in this highly vulnerable population. OBJECTIVE The objective of the present study was to evaluate the cervicovaginal levels of the proinflammatory cytokines IL-1 beta, IL-6 and IL-8 and bacterial sialidase in pregnant adolescents with bacterial vaginosis. METHODS Between 2009 and 2011, a total of 168 pregnant adolescents were recruited for this cross-sectional study in outpatient clinics for obstetric care for adolescents in the metropolitan area of Belém, Pará, northern region of Brazil. The ethics committee of Universidade Federal do Pará (#002/2009) approved the study. All the participants were accompanied by a family member or partner who was older than 18 years and signed a written consent statement. The inclusion criteria were that the patient needed to be pregnant, younger than 19 years and HIV-negative; and to have not used antibiotics recently (last 30 days), to have not had sexual intercourse over the last three days and to have undergone vaginal ultrasound examination within the last three days. All the adolescents answered a standardized questionnaire in order to obtain sociodemographic and behavioral data and underwent pelvic examination for cervicovaginal sampling. After inserting a non-moisturized speculum, vaginal ph was ascertained using commercial ph strips (Merck, Darmstadt, Germany) and the whiff test, performed by adding 10% KOH (potassium hydroxide) to the samples. Swabs were taken from the vaginal wall and smeared on microscope slides for Gram staining and classification of the vaginal flora in accordance with the scoring system proposed by Nugent et al., 20 as normal (0-3), intermediate (4-6) or bacterial vaginosis (7-10). The diagnosis of bacterial vaginosis was based on Nugent scores, regardless of the vaginal ph and whiff test. Gram-stained smears were also used for diagnosing candidiasis when Candida sp. hyphae were detected. Additionally, samples taken from the vaginal vault were cultured in Diamond s liquid medium for detection of Trichomonas vaginalis. Endocervical samples were also obtained for Neisseria gonorrhoeae and Chlamydia trachomatis detection by means of, respectively, culturing in Thayer-Martin and the polymerase chain reaction (PCR). Cultures for Neisseria gonorrhoeae were incubated at 37 C for up to 48 hours. For Chlamydia trachomatis PCR, two sets of primers were used: CTP1 (5 -TAG TAA CTG CCA CTT CAT CA-3 ) and CTP2 (5 -TTC CCC TTG TAA TTC GTT GC-3 ); or PL6.1 (5 -AGA GTA CAT CGG TCA ACG A-3 ) and PL62 (5 -TCA CAG CGG TTG CTC GAA GCA-3 ), 21 resulting in product sizes of 201 bp and 130 bp, respectively. Reactions were performed using GoTaq Green Master Mix (Promega, Madison, WI, USA) and 4 µl of DNA template with the following cycling: 95 C for 5 min followed by 40 repetitions of 95 C for 1 min, 55 C for 1 min and 72 C for 1.5 min, and finally, 72 C for 5 min (Mastercycler, Eppendorf, Germany). Cervicovaginal lavages were performed using 3 ml of sterile 0.9% saline solution. The total volume was recovered using a plastic pipette, immediately refrigerated and then transported to the laboratory within 4 hours. After centrifugation at 800 x g for 10 minutes, the supernatant was used in the enzyme-linked immunosorbent assay (ELISA) to determine the levels of the proinflammatory cytokines IL-1 beta, IL-6 and IL-8 (DuoSet Kits, R&D Systems, Minneapolis, MN, USA), in accordance with the 466 Sao Paulo Med J. 2015; 133(6):465-70

15 Bacterial vaginosis in pregnant adolescents: proinflammatory cytokine and bacterial sialidase profile. Cross-sectional study ORIGINAL ARTICLE manufacturer s instructions. Supernatants of the lavages were also used to determine the sialidase levels by means of conversion of the fluorogenic substrate 2-(4-methylumbelliferyl)-α-D-Nacetylneuraminic acid (MUAN) (Sigma-Aldrich, St. Louis, MO, USA), in accordance with methods detailed previously. 13,15 In both the cytokine and the sialidase assays, samples were tested in duplicates and, if the measured concentration was above the standard curve, the samples were diluted and retested. The intra and interassay variability rates were < 11.0%. The minimum detectable levels for IL-1 beta, IL-6, IL-8 and sialidase assays were, respectively, 0.1 pg/ml, 1.1 pg/ml, 15.9 pg/ml and 0.2 ng/ml. Out of the 168 adolescents initially recruited, those who tested positive for C. trachomatis (n = 28; 16.7%), trichomoniasis (n = 5; 3.0%) or candidiasis (n = 12; 7.1%) or presented intermediate flora (n = 3; 1.8%) were excluded from the analysis. The remaining 120 adolescents who presented all the criteria for enrollment were divided into two groups according to the classification of their vaginal flora as normal (n = 68; 56.7%) or bacterial vaginosis (n = 52; 43.3%). Comparisons of discrete and continuous variables between the groups were made respectively using the chi-square test or Fisher s exact test and the nonparametric Mann-Whitney test. Cytokine and sialidase levels were compared using the Mann- Whitney test. All analyses were performed using the GraphPad Prism 5.0 software (GraphPad, CA, USA), considering P < 0.05 to be statistically significant. Although no minimum sample size was calculated during the study design phase, a post-hoc analysis showed that the sample had a minimum test power of 85%, considering the IL-1 beta and IL-8 levels found in the cervicovaginal samples from subjects with normal flora and bacterial vaginosis. RESULTS The sociodemographic, behavioral and clinical characteristics of the 120 adolescents enrolled are shown in Table 1. Most of them reported that they were married or cohabiting with a partner (n = 64/102; 62.7%), did not have a paid job (n = 90/100; 90.0%) and had not had any STI diagnosed previously (n = 80/87; 92.0%). The gestational age at enrollment and all the other characteristics did not differ between the groups (P > 0.05) except for vaginal ph (P < ) and the number of whiff-positive samples (P < ), which were significantly higher in individuals with bacterial vaginosis than in those with normal flora. The results from cytokine assays, with the measured levels of IL-1 beta, IL-6 and IL-8 are shown in Figure 1. The IL-1 beta level was above the detection limit of the assay in 96 cervicovaginal samples (80.0%), while IL-6 and IL-8 levels were determined in respectively 42 (35.0%) and 111 (92.5%) of the 120 samples. The pregnant adolescents with bacterial vaginosis had higher IL-1 beta levels (median pg/ml; range ) in the Table 1. Sociodemographic, behavioral and clinical characteristics of the 120 pregnant adolescents included in the study. Values shown as median (range) or n/total number (%) Characteristics* Normal (n = 68) Bacterial vaginosis (n = 52) P Age (years) 16 (13-19) 16 (13-19) 0.73 Gestational age 18 weeks 1 day (8 weeks - 4 weeks 1 day) 16 weeks 1 day (7 weeks - 35 weeks 2 days) cervicovaginal samples than did those with normal vaginal flora (median 4.3 pg/ml; range ) (P < ). IL-6 levels were also significantly higher in individuals with bacterial vaginosis (median 0.0 pg/ml; range ) than in those with normal flora (median 0.0 pg/ml; range ) (P = 0.03). Similarly to IL-1 beta and IL-6, the IL-8 levels were higher in individuals with bacterial vaginosis (median pg/ml; range ) than in those with normal flora (median pg/ml; range ) (P = 0.002). Bacterial sialidase was not detected in samples from pregnant adolescents with normal flora. However, a total of 35 (67.2%) of the 52 adolescents with bacterial vaginosis had detectable cervicovaginal levels of this enzyme, with a median concentration of 10.3 ng/ml (range ng/ml). DISCUSSION In this cross-sectional study, we determined the cervicovaginal levels of proinflammatory cytokines and sialidase in pregnant adolescents with bacterial vaginosis living in the northern region of Brazil and compared these levels with those with normal lactobacilli-dominated flora. To our knowledge, this is the first report on the inflammatory response to bacterial vaginosis among pregnant adolescents. We are aware of the limitation that this study presents through not having an initial estimate for the sample size. However, posthoc analysis produced a satisfactory power test based on the levels of the two cytokines quantified. Therefore, we believe that this limitation does not hamper the findings shown here Marital status Single 19/57 (33.3) 19/45 (42.2) Married or cohabiting 38/57 (66.7) 26/45 (57.8) 0.36 Education (years at school) 6.5 (0-12) 7 (1-11) 0.88 Full or part-time paid job 5/56 (8.9) 5/44 (11.4) 0.74 History of previous STI 5/55 (9.1) 2/32 (6.2) 1.00 Regular use of condoms 20/56 (35.7) 16/42 (38.1) 1.00 Vaginal ph 4.4 ( ) 5,0 ( ) < Positive whiff test 8/54 (14.8) 39/43 (90.7) < *Total number of women may vary among the categories, since some of the data were unavailable or the patient refused to answer; STI = sexually transmitted infection; Mann-Whitney test; chi-square test; Fisher exact test; P < Sao Paulo Med J. 2015; 133(6):

16 ORIGINAL ARTICLE Ferreira CST, Marconi C, Parada CMLG, Cassamassimo Duarte MT, Gonçalves APO, Rudge MVC, Silva MG A B C Interleukin 1 beta (pg/ml) Interleukin 6 (pg/ml) Interleukin 8 (pg/ml) Recent studies have pointed out that vaginal immunity is compromised by disrupted vaginal flora, thereby leading to higher susceptibility to STI acquisition and transmission. 7,22 Another noteworthy finding is that adolescents have vaginal immunityrelated mediators with a composition that differs from that of adults mediators and consequently may respond differently to Normal Normal Normal P < P = 0.03 P = Figure 1. Levels of the proinflammatory cytokines interleukin 1 beta (A), interleukin 6 (B) and interleukin 9 (C) in the cervicovaginal samples from 68 pregnant adolescents with bacterial vaginosis (BV) and 52 with normal vaginal flora. Horizontal bars represent the median (pg/ml). BV BV BV changes in the composition of the vaginal flora. 12 In the present study, all cervicovaginal infections and alterations other than bacterial vaginosis were excluded, since they could act as potential confounders for cervicovaginal cytokine and sialidase levels. 23,24 Our population was homogeneous between the two study groups, not only regarding sociodemographic variables but also in relation to the gestational age at the time of enrollment. It has been consistently shown in the literature that cervicovaginal IL-1 beta levels increase in response to bacterial vaginosis in non-pregnant 3,15,25 and pregnant women, 16 which is in agreement with our current data on pregnant adolescents. IL-1 beta is crucial for the initial immune response to pathogens, but higher levels of this cytokine in the vaginal milieu increase the vulnerability to STI acquisition. 8 Non-pregnant adolescents have significantly higher IL-1 beta levels, and this has been proposed as one of the mechanisms involved in the higher prevalence of STIs in this population. 12 Data on vaginal IL-6 in the literature have shown that there is an association between increased levels of this cytokine in situations of aerobic vaginitis, but not of bacterial vaginosis. 13,24 However, it has already been demonstrated that IL-6 levels are higher in pregnant women with bacterial vaginosis than in non-pregnant women with bacterial vaginosis. 16 Recently, Balkus et al. 10 failed to demonstrate any independent association between increased vaginal IL-6 levels and either pregnancy or bacterial vaginosis. However, they found that non-pregnant adolescents had higher IL-6 levels than those of adults. 12 Our data provide the new information that IL-6 levels are even higher in pregnant adolescents with bacterial vaginosis. Normally, IL-8 levels do not increase in situations of bacterial vaginosis, which explains the unchanged number of leukocytes in vaginal smears, in comparison with normal flora. 26 On the other hand, this scenario changes when pregnant women with bacterial vaginosis are evaluated in relation to non-pregnant women with the same floral alterations, such that significantly higher IL-8 levels are seen in pregnancy. 16 According to Balkus et al., increased IL-8 levels were independently associated with both pregnancy and bacterial vaginosis. 10 No change in cervicovaginal IL-8 levels has been associated with adolescence so far. 12 Thus, based on data on the literature, we suggest that the increased cervicovaginal IL-8 levels reported here may have been due to pregnancy status rather than the younger age of the population. Bacterial sialidase is produced by several microorganisms associated with bacterial vaginosis 27 and it degrades local immunoglobulins, thereby leading to impairment of the local immune response. 18 In agreement with previous findings, the present study showed that sialidase was mostly detectable in situations of abnormal vaginal flora. This finding is particularly important 468 Sao Paulo Med J. 2015; 133(6):465-70

17 Bacterial vaginosis in pregnant adolescents: proinflammatory cytokine and bacterial sialidase profile. Cross-sectional study ORIGINAL ARTICLE in pregnancy, since detection of sialidase on vaginal samples has already been correlated with serious obstetric complications, such as preterm birth and preterm premature rupture of membranes Moreover, higher vaginal sialidase levels may increase the vulnerability to STIs during bacterial vaginosis, since they compromise the local defense against pathogens. 18 The current findings highlight the importance of conducting further studies aimed at achieving better understanding of the vaginal immunity of adolescents. Considering that adequate treatment of bacterial vaginosis may be beneficial to this population by preventing STI acquisition and the risk of poor pregnancy outcomes, screening programs should be included during antenatal care. Further studies are still needed in order to develop and implement new strategies for STI prevention and for improvement of the reproductive health of this population. The high rates of abnormal vaginal flora and cervicovaginal infections reported here reinforce the need for screening for this particularly vulnerable population at prenatal services. CONCLUSION Bacterial vaginosis is associated with a pronounced local inflammatory response and increased sialidase levels in pregnant adolescents, thereby contributing towards higher susceptibility of this population to STIs. REFERENCES 1. Hillier SL, Nugent RP, Eschenbach DA, et al. Association between bacterial vaginosis and preterm delivery of a low-birth-weight infant. The Vaginal Infections and Prematurity Study Group. N Engl J Med. 1995;333(26): Giraldo PC, Araújo ED, Junior JE, et al. The prevalence of urogenital infections in pregnant women experiencing preterm and full-term labor. Infect Dis Obstet Gynecol. 2012;2012: Brabin L, Fairbrother E, Mandal D, et al. Biological and hormonal markers of chlamydia, human papillomavirus, and bacterial vaginosis among adolescents attending genitourinary medicine clinics. Sex Transm Infect. 2005;81(2): Holst E, Wathne B, Hovelius B, Mårdh PA. Bacterial vaginosis: microbiological and clinical findings. Eur J Clin Microbiol. 1987;6(5): Hillier SL, Martius J, Krohn M, et al. A case-control study of chorioamnionic infection and histologic chorioamnionitis in prematurity. N Engl J Med. 1988;319(15): Gravett MG, Hummel D, Eschenbach DA, Holmes KK. Preterm labor associated with subclinical amniotic fluid infection and with bacterial vaginosis. Obstet Gynecol. 1986;67(2): Sewankambo N, Gray RH, Wawer MJ, et al. HIV-1 infection associated with abnormal vaginal flora morphology and bacterial vaginosis. Lancet. 1997;350(9077): Sturm-Ramirez K, Gaye-Diallo A, Eisen G, Mboup S, Kanki PJ. High levels of tumor necrosis factor-alpha and interleukin-1beta in bacterial vaginosis may increase susceptibility to human immunodeficiency virus. J Infect Dis. 2000;182(2): Lockwood CJ, Ghidini A, Wein R, et al. Increased interleukin-6 concentrations in cervical secretions are associated with preterm delivery. Am J Obstet Gynecol. 1994;171(4): Balkus J, Agnew K, Lawler R, Mitchell C, Hitti J. Effects of pregnancy and bacterial vaginosis on proinflammatory cytokine and secretory leukocyte protease inhibitor concentrations in vaginal secretions. J Pregnancy. 2010;2010: Anderson BL, Mendez-Figueroa H, Dahlke JD, et al. Pregnancyinduced changes in immune protection of the genital tract: defining normal. Am J Obstet Gynecol. 2013;208(4):321.e Madan RP, Carpenter C, Fiedler T, et al. Altered biomarkers of mucosal immunity and reduced vaginal Lactobacillus concentrations in sexually active female adolescents. PLoS One. 2012;7(7):e Marconi C, Donders GG, Bellen G, et al. Sialidase activity in aerobic vaginitis is equal to levels during bacterial vaginosis. Eur J Obstet Gynecol Reprod Biol. 2013;167(2): Cauci S. Vaginal Immunity in Bacterial Vaginosis. Curr Infect Dis Rep. 2004;6(6): Marconi C, Donders GG, Parada CM, Giraldo PC, da Silva MG. Do Atopobium vaginae, Megasphaera sp. and Leptotrichia sp. change the local innate immune response and sialidase activity in bacterial vaginosis? Sex Transm Infect. 2013;89(2): Beigi RH, Yudin MH, Cosentino L, Meyn LA, Hillier SL. Cytokines, pregnancy, and bacterial vaginosis: comparison of levels of cervical cytokines in pregnant and nonpregnant women with bacterial vaginosis. J Infect Dis. 2007;196(9): Workowski KA, Berman S; Centers for Disease Control and Prevention (CDC). Sexually transmitted diseases treatment guidelines, MMWR Recomm Rep. 2010;59(RR-12): Lewis WG, Robinson LS, Perry J, et al. Hydrolysis of secreted sialoglycoprotein immunoglobulin A (IgA) in ex vivo and biochemical models of bacterial vaginosis. J Biol Chem. 2012;287(3): Cauci S, Culhane JF. High sialidase levels increase preterm birth risk among women who are bacterial vaginosis-positive in early gestation. Am J Obstet Gynecol. 2011;204(2):142.e Nugent RP, Krohn MA, Hillier SL. Reliability of diagnosing bacterial vaginosis is improved by a standardized method of gram stain interpretation. J Clin Microbiol. 1991;29(2): Morré SA, Sillekens P, Jacobs MV, et al. RNA amplification by nucleic acid sequence-based amplification with an internal standard enables reliable detection of Chlamydia trachomatis in cervical scrapings and urine samples. J Clin Microbiol. 1996;34(12): Cohen CR, Lingappa JR, Baeten JM, et al. Bacterial vaginosis associated with increased risk of female-to-male HIV-1 transmission: a prospective cohort analysis among African couples. PLoS Med. 2012;9(6):e Sao Paulo Med J. 2015; 133(6):

18 ORIGINAL ARTICLE Ferreira CST, Marconi C, Parada CMLG, Cassamassimo Duarte MT, Gonçalves APO, Rudge MVC, Silva MG 23. Cauci S, Culhane JF. Modulation of vaginal immune response among pregnant women with bacterial vaginosis by Trichomonas vaginalis, Chlamydia trachomatis, Neisseria gonorrhoeae, and yeast. Am J Obstet Gynecol. 2007;196(2):133.e Donders GG, Vereecken A, Bosmans E, et al. Definition of a type of abnormal vaginal flora that is distinct from bacterial vaginosis: aerobic vaginitis. BJOG. 2002;109(1): Hedges SR, Barrientes F, Desmond RA, Schwebke JR. Local and systemic cytokine levels in relation to changes in vaginal flora. J Infect Dis. 2006;193(4): Cauci S, Guaschino S, De Aloysio D, et al. Interrelationships of interleukin-8 with interleukin-1beta and neutrophils in vaginal fluid of healthy and bacterial vaginosis positive women. Mol Hum Reprod. 2003;9(1): McGregor JA, French JI, Jones W, et al. Bacterial vaginosis is associated with prematurity and vaginal fluid mucinase and sialidase: results of a controlled trial of topical clindamycin cream. Am J Obstet Gynecol. 1994;170(4): ; discussion Cauci S, Hitti J, Noonan C, et al. Vaginal hydrolytic enzymes, immunoglobulin A against Gardnerella vaginalis toxin, and risk of early preterm birth among women in preterm labor with bacterial vaginosis or intermediate flora. Am J Obstet Gynecol. 2002;187(4): The results from this study were presented at the Ninth Congress of the Brazilian Society of Sexually Transmitted Diseases and Fifth Brazilian Congress on AIDS, which took place in Salvador, Bahia, Brazil, from August 18 to 21, 2013 Source of funding: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Grants: #551245/ and # [2011-1] Conflict of interests: None Date of first submission: June 27, 2014 Last received: October 21, 2014 Accepted: October 27, 2014 Address for correspondence: Márcia Guimarães da Silva Departamento de Patologia Faculdade de Medicina de Botucatu (FMB) Universidade Estadual Paulista (Unesp) Distrito de Rubião Júnior, s/n o Botucatu (SP) Brasil CEP Tel. (+55 14) Fax. (+55 14) mgsilva@fmb.unesp.br 470 Sao Paulo Med J. 2015; 133(6):465-70

19 DOI: / ORIGINAL ARTICLE Patients perceptions about diagnosis and treatment of chronic myeloid leukemia: a cross-sectional study among Brazilian patients Percepções dos pacientes sobre diagnóstico e tratamento da leucemia mieloide crônica: estudo transversal entre pacientes brasileiros Nelson Hamerschlak I, Carmino de Souza II, Ana Lúcia Cornacchioni III, Ricardo Pasquini IV, Daniel Tabak V, Nelson Spector VI, Merula Steagall VII Department of Hematology, Hospital Israelita Albert Einstein (HAIE) and Associação Brasileira de Linfoma e Leucemia (Abrale), São Paulo, Brazil I MD, PhD. Head of Bone Marrow Transplantation Unit, Hospital Israelita Albert Einstein (HIAE) and Associação Brasileira de Linfoma e Leucemia (ABRALE), São Paulo, Brazil. II MD, PhD. Titular Professor of Internal Medicine, Universidade Estadual de Campinas (Unicamp), Campinas and Associação Brasileira de Linfoma e Leucemia (ABRALE), São Paulo, Brazil. III MD. Attending Physician, Instituto de Tratamento do Câncer Infantil (ITACI), Instituto da Criança (ICR), Hospital de Clínicas da Universidade de São Paulo (HC-USP) and Associação Brasileira de Linfoma e Leucemia (ABRALE), São Paulo, Brazil. IV MD, PhD. Senior Professor, Postgraduate Program on Internal Medicine, Universidade Federal do Paraná (UFP), Paraná and Associação Brasileira de Linfoma e Leucemia (ABRALE), São Paulo, Brazil. V MD, PhD. Director, Centro de Tratamento Oncológico (Centron), São Paulo and Associação Brasileira de Linfoma e Leucemia (ABRALE), São Paulo, Brazil. VI MD, PhD. Titular Professor, Universidade Federal do Rio de Janeiro (UFRJ), and Head of the Hematology Service, Hospital Universitário Clementino Fraga Filho (HUCCFF), Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro; and Associação Brasileira de Linfoma e Leucemia (ABRALE), São Paulo, Brazil. VII President, Associação Brasileira de Linfoma e Leucemia (ABRALE), São Paulo, Brazil. KEY WORDS: Leukemia, myelogenous, chronic, BCR-ABL positive. Leukemia, myeloid, chronic-phase. Protein-tyrosine kinases. National health programs. Perception. Patient compliance. PALAVRAS-CHAVE: Leucemia mielogênica crônica BCR-ABL positiva. Leucemia mieloide de fase crônica. Proteínas tirosina quinases. Programas nacionais de saúde. Percepção. Cooperação do paciente. ABSTRACT CONTEXT AND OBJECTIVES: Chronic myeloid leukemia (CML) requires strict daily compliance with oral medication and regular blood and bone marrow control tests. The objective was to evaluate CML patients perceptions about the disease, their access to information regarding the diagnosis, monitoring and treatment, adverse effects and associations of these variables with patients demographics, region and healthcare access. DESIGN AND SETTING: Prospective cross-sectional study among CML patients registered with the Brazilian Lymphoma and Leukemia Association (ABRALE). METHODS: CML patients receiving treatment through the public healthcare system were interviewed by telephone. RESULTS: Among 1,102 patients interviewed, the symptoms most frequently leading them to seek medical care were weakness or fatigue. One third were diagnosed by means of routine tests. The time that elapsed between first symptoms and seeking medical care was ± days. Most patients had been tested at least once for Philadelphia chromosome, but 43.2% did not know the results. 64.8% had had polymerase chain reaction testing for the BCR/ABL gene every three months. 47% believed that CML could be controlled, but 33.1% believed that there was no treatment. About 24% reported occasionally stopping their medication. Imatinib was associated with nausea, cramps and muscle pain. Self-reported treatment adherence was significantly associated with normalized blood count, and positively associated with imatinib. CONCLUSIONS: There is a lack of information or understanding about disease monitoring tools among Brazilian CML patients; they are diagnosed quickly and have good access to treatment. Correct comprehension of CML control tools is impaired in Brazilian patients. RESUMO CONTEXTO E OBJETIVOS: Leucemia mieloide crônica (CML) exige estrita adesão à medicação oral e ao monitoramento do sangue e da medula. O objetivo foi avaliar percepções de pacientes com leucemia mieloide crônica (LMC) sobre a doença, seu acesso à informação sobre diagnóstico, monitoramento e tratamento, efeitos adversos e a associação destes com dados demográficos, geográficos e de acesso a tratamento. DESENHO E LOCAL: Estudo prospectivo transversal realizado com pacientes de LMC cadastrados na Associação Brasileira de Leucemia e Linfoma (Abrale). MÉTODOS: Pacientes com LMC recebendo tratamento do sistema público de saúde foram entrevistados por telefone. RESULTADOS: Entre os pacientes entrevistados, os sintomas mais frequentemente levando à busca de consulta foram fraqueza e fadiga. Um terço foi diagnosticado por exames de rotina. O tempo entre sintoma inicial e procura por ajuda foi de 42,28 ± 154,21 dias. A maioria foi testada pelo menos uma vez para o cromossomo Filadélfia, mas 43,2% não sabiam os resultados. 64,8% fizeram exame de reação em cadeia da polimerase para o gene BCR/ABL a cada três meses. 47% acreditavam que LMC pode ser controlada, mas 33,1% acham que não há tratamento. Cerca de 24% disseram que ocasionalmente interrompem o tratamento. Imatinibe associou-se com náusea, câimbra e dor muscular. Aderência auto-reportada associou-se significativamente com hemograma normal e positivamente com uso de imatinibe. CONCLUSÕES: Falta informação ou compreensão sobre monitoramento entre pacientes com LMC; eles recebem diagnóstico rapidamente e têm bom acesso ao tratamento. A correta compreensão das ferramentas de controle em LMC está prejudicada entre eles. Sao Paulo Med J. 2015; 133(6):

20 ORIGINAL ARTICLE Hamerschlak N, Souza C, Cornacchioni AL, Pasquini R, Tabak D, Spector N, Steagall M INTRODUCTION Treatment of chronic myeloid leukemia (CML) used to include bone marrow transplantation, hydroxyurea and therapeutic regimens based on interferon-alpha (IFN-alpha). About 10 years ago, imatinib mesylate, a derivative of phenyl-2-amino-pyrimidine that is a selective inhibitor of BCR-ABL tyrosine kinase, which induces hematological and cytogenetic remission in CML cases, became the treatment of choice. 1 The average age of 50 years among patients originally affected by CML (median of 60), as well as the lack of histocompatible donors and the early and late risks, limits the option of bone marrow transplantation to a minority of patients. 2-4 Imatinib mesylate is a drug with proven efficacy for treating patients with CML and is indicated as firstline medication for patients with Philadelphia chromosome (Ph+) positive CML (a chromosomal translocation associated with CML that is used in diagnosing the disease). 5,6 Today, secondgeneration tyrosine kinase inhibitors such as dasatinib and nilotinib have also been shown to be efficacious as first-line therapy Every Brazilian citizen has the right to receive imatinib mesylate for treatment of CML, on a cost-free basis, provided by the government. However, no study has investigated the access to these treatments in Brazil yet. This chronic disease may cause significant changes to the daily lives of individuals and their families, 11,12 since management of this disease involves strict daily compliance with oral medication and regular blood and bone marrow control examinations. 13,14 In the context of chronic diseases, cancer is seen in the popular imagination as a cause of rapid finitude and suffering, among other meanings. Although there are cultural differences in how the disease and patients expectations regarding the physician s role are viewed, 15 even without universal policies to inform and involve patients in decisions that affect their care, 16 most patients want information about their diagnosis and want their families to also be informed, even when their illnesses are severe This has proved to be an important therapeutic tool, 13,21 and some authors have suggested that this information decreases the patient s sense of isolation and contributes towards mutual cooperation in the doctor-patient relationship. 21 Hematology-oncology is a specialty that deals with patients who now can count on increased survival based on the proposed treatments treatments that, of course, are not without risks or side effects, but are tolerable. However, few studies have been conducted in Brazil to determine patients desire to participate in treatment decisions, and there have not been any studies specifically on CML patients that have assessed their access to treatment and the time required for a diagnosis to be reached. Since there may be significant differences in patients perceptions of the quality and quantity of information received and in their participation in medical decisions among populations in different countries, it is important that studies like this should be conducted in Brazil. 17 OBJECTIVE The purpose of this study was to evaluate Brazilian CML patients perceptions about the disease, their access to information regarding the diagnosis, treatment and care received, adverse effects and relationships with doctors; and associations of these variables with patients demographics, region and access to healthcare. METHODS Design, participants and location This was a prospective, longitudinal study among patients with CML who were registered with the Brazilian Lymphoma and Leukemia Association (ABRALE). ABRALE is a civil society organization formed by patients with leukemia and lymphoma and their families, which provides information and support to patients, and educational programs, publications and events to healthcare professionals across the country. The study was approved by the Ethics Committee of the Albert Einstein Institute for Teaching and Research. During the period between April 28, 2008, and February 8, 2010, all patients registered with ABRALE were surveyed by telephone about the care they had received for this disease. Patients were enrolled if they had a diagnosis of CML and were registered in ABRALE. Patients are registered with ABRALE through direct enrolment in the healthcare services where they are treated, in eight Brazilian state capitals (Rio de Janeiro, Curitiba, Goiânia, Recife, Porto Alegre, Belo Horizonte, São Paulo and Salvador) and in the city of Campinas (state of São Paulo). All the participants were interviewed 1 to 3 times, and were asked about their perceptions of the disease and its treatment, the drugs they were using and any adverse effects, and their relationships with their doctors. A questionnaire was specially designed for this study, including questions concerning the variables below. Patients registered with ABRALE, i.e. patients undergoing treatment for CML who were receiving drugs through the Brazilian public healthcare system (SUS, Sistema Único de Saúde), were included. The telephone number in ABRALE S records was used to contact patients as many times as needed until they had time to be interviewed. Deceased patients and those whose contact information in the database was out of date or incorrect were excluded. If patients could not or did not want to participate in an interview at the first contact (because they were busy or not feeling well), they were called at least one more time. Sometimes, they were called more than three times. If in the end they could not be reached, they were excluded from the study. Variables studied The patients demographic characteristics, such as age, education level, geographic location and access to healthcare services were checked. The symptoms that led patients to seek healthcare, their specialists, the tests that were requested and performed 472 Sao Paulo Med J. 2015; 133(6):471-9