Metabolic abnormalities and liver stiffness evaluated by Transient Elastography in HIV and HIV/HCV coinfected patients

2013/2014 Catarina Magalhães Castelo Branco Metabolic abnormalities and liver stiffness evaluated by Transient Elastography in HIV and HIV/HCV coinfected patients março, 2014 Mestrado Integrado em Medicina Área: Endocrinologia Trabalho efetuado sob a Orientação de: Doutora Paula Isabel Marques Simões de Freitas Trabalho organizado de acordo com as normas da revista: BMC Infectious Diseases Catarina Magalhães Castelo Branco Metabolic abnormalities and liver stiffness evaluated by Transient Elastography in HIV and HIV/HCV coinfected patients Março, 2014 - 1 - Metabolic abnormalities and liver stiffness evaluated by Transient Elastography in HIV and HIV/HCV coinfected patients Catarina Castelo-Branco1§, Eva Lau1,2, Joana Oliveira1,2, Ana Cristina Santos1,3, Rosário Serrão4, Luís Ferreira-Pinto1, António Sarmento1,4, Davide Carvalho1,2, Paula Freitas1,2 1University of Porto Medical School 2Department of Endocrinology, Diabetes and Metabolism of Centro Hospitalar São João, E.P.E. 3Department of Clinical Epidemiology, Predictive Medicine and Public Health , University of Porto Medical School, and University of Porto Institute of Public Health, Porto, Portugal 4 Infectious Diseases Department of Centro Hospitalar de São João, E.P.E. §Corresponding author Email addresses: CCB: [email protected] - 2 - Abstract Background Hepatitis C Virus (HCV) infection is highly prevalent among HIV infected patients. Liver disease and metabolic complications are the major concerns about their management and the main causes of morbimortality. It is still not fully understood how the presence of coinfection affects the metabolic profile and the natural history of the liver disease. Methods Retrospective cohort study including 299 adults under combined antiretroviral therapy (cART) (236 HIV monoinfected and 63 HIV/HCV coinfected patients). Insulin Resistance (IR) was defined as HOMA-IR≥2. Coinfected patients were divided into 4 groups according to the presence of fibrosis defined by Liver Stiffness (LS): 1) absence of significant fibrosis, if LS≤6.0 kPa; 2) undetermined fibrosis, if 6.0<LS<9.0 kPa; 3) significant fibrosis, if 9.0≤LS<14.6 kPa; 4) cirrhosis, if LS≥14.6 kPa. Results HIV/HCV coinfected patients presented lower body mass index, waist circumference, hypertension and lipid levels in comparison to HIV mono-infected patients. Glucose metabolism parameters did not differ according to HCV serostatus and IR was found in approximately half of the patients in both groups. In HIV/HCV coinfected patients, fibrosis grade was associated with differences in nonnucleoside reverse transcriptase inhibitors (NNRTI) use, duration of HCV infection and HCV genotype. Regarding metabolic characteristics, higher glutamic oxaloacetic transaminase (GOT) levels and lower total cholesterol (TC), non-high density lipoprotein cholesterol (Non-HDL-C) and albumin were observed in the presence of liver fibrosis. - 3 - NNRTI, TC, GOT(ln), albumin, glucose(ln) and age were included in the linear regression for LS. TC [β=-0.038 (-0.074;-0.001)] and albumin [β=-0.896 (-1.191;- 0.602)] were found to be inversely associated with LS. Conclusions HIV/HCV coinfection was associated with better metabolic profile than HIV monoinfection, except for insulin resistance that was similar in both groups. Total cholesterol and albumin were independently and inversely associated with liver stiffness. Keywords HIV; HCV; Coinfection; Liver stiffness; Liver fibrosis; Insulin resistance; HOMA; Dyslipidemia Background Hepatitis C Virus infection has an extremely high prevalence among Human Immunodeficiency Virus (HIV) infected patients, affecting 4 to 5 million people worldwide [1]. In the setting of HIV/HCV coinfection, rapid rates of fibrosis progression were observed [2] and end-stage liver disease is now the primary cause of death among patients under cART [3]. HIV/HCV coinfection pathogenesis is still not fully understood. Both HIV and HCV infections give rise to glucose and lipid disorders, bringing up to discussion how the presence of coinfection influences the metabolic status. Metabolic changes in HIV/HCV coinfected patients have been associated with accelerated progression of liver - 4 - disease, lower rates of response to anti-HCV therapy as well as cardio and cerebrovascular complications [4, 5]. As a result, the development of measures that may slow down the progression of HCV related liver disease should be a priority [3]. Earlier recognition of these metabolic changes may be an important step to allow an improved therapeutic approach, both to the viral infection and to the metabolic diseases [6]. Staging and monitoring liver fibrosis is a key concern in the management of coinfected patient, which requires a tool that is reliable, relatively inexpensive and accurate. Transient Elastography (TE) is a non-invasive test to assess liver fibrosis by measuring liver stiffness, validated to predict significant fibrosis and cirrhosis in HIV/HCV coinfected patients. Besides avoiding the complications inherent to liver biopsy, it also outgrows the latter in terms of sampling errors, inter-observer variability and acceptance by the patients [7, 8]. The aim of this study was to evaluate the prevalence of clinical and metabolic parameters, comparing HIV monoinfected with HIV/HCV coinfected patients and to identify the factors associated with significant liver fibrosis, assessed by TE in HIV/HCV coinfected patients. Methods Subjects As part of a cross-sectional study, 299 non-institutionalized HIV infected Caucasian adults under cART followed in the Infectious Diseases and Endocrinology Out-patient Clinic of Centro Hospitalar de São João were included: 236 HIV monoinfected and 63 HIV/HCV coinfected patients. Only the coinfected patients who had a TE evaluation from 2013 were included. Patients with a previous diagnosis of - 5 - diabetes or hepatitis B were excluded. The study protocol was approved by the Hospital Ethics Committee and all patients provided informed consent. Clinical assessment For each patient, the following information was collected using a standardized protocol: demographic data (age, gender), duration of HIV and HCV infection, duration of cART, history of hypertension, use of antihypertensive or lipid lowering drugs and alcohol use. Weight, height, waist circumference and resting blood pressure were measured as previously described [9]. A venous blood sample was drawn after a 12-hour overnight fast. All the samples were analyzed at the central laboratory of our hospital. Plasma glucose, insulin, GOT, glutamic pyruvic transaminase (GPT), gamma-glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), albumin, platelets, total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and triglycerides (TGL) were determined using automatic standard routine enzymatic methods. The CD4+ cell count was determined by flow cytometry and plasma RNA-HIV and RNA-HCV were measured by a quantitative reverse transcriptase polymerase chain reaction (PCR), which had a lower limit of detection of 50 copies/mL. HCV infection was diagnosed by the presence of serum antibodies against HCV (RIBA test) and detectable serum HCV RNA PCR. Insulin resistance Insulin resistance was defined by the homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity by the quantitative insulin sensitivity check index (QUICKI). These indexes were calculated by the following formulas: HOMA-IR index = (fasting plasma insulin [mU/L] x fasting plasma glucose [mg/dL])/450 [10]. QUICKI = 1/[log (fasting insulin [mU/L]) +log (fasting plasma glucose [mg/dL]) - 12 - immune response both in HIV and HCV infections give rise to glucose and lipid metabolic disorders that, in turn, are risk factors for liver damage [1, 28]. cART benefits in reducing liver disease progression by improving host immunity seem to outweigh its hepatotoxicity and IR risk [3, 29, 30]. In HCV infection, low lipid levels have been correlated not only with steatosis and more advanced liver fibrosis, but also with non-response to interferon treatment [4]. This is in line with what was observed in this study: those with higher grades of fibrosis had lower TC and non-HDL-C. TC remained significantly inversely associated with LS, independently of age, NNRTI, TC, GOT and albumin. GOT elevations are common with fibrosis progression but they are not a good marker, especially in those with HIV infection, in whom the use of cART also contributes to alterations in the hepatic profile. Normal level of GOT should not exclude the need of anti-HCV treatment because there are patients who develop fibrosis in the absence of transaminases elevation [25]. Lower albumin is an established marker of hepatic insufficiency. In our cohort, albumin levels were negatively associated with LS in our model for liver fibrosis, independently of the other variables included in the multivariated model. Current literature is not yet accordant regarding IR importance to the pathogenesis of accelerated fibrosis progression in HIV/HCV infected individuals, as some studies described a positive correlation between IR and more advanced grades of fibrosis [12, 31, 32] and others did not find that association [13, 33]. Moreover, it was reported that IR is associated with a poor sustained virological response to HCV treatment in HIV/HCV coinfected as well as in HCV monoinfected patients [34, 35]. On the other hand, this relationship has been consistently reported for HCV monoinfection, suggesting that IR is a determinant of fibrogenesis in chronic hepatitis - 13 - C, independent of hepatic steatosis, through direct stimulation of hepatic stellate cells by hyperglycaemia and hyperinsulinemia to proliferate and to secrete extracellular matrix [36-39]. Concomitantly, IR promotes hepatic lipogenesis and it is plausible that steatosis per se contributes to fibrogenesis. No association was found for glucose (ln) and LS in the multivariated model, similarly to what has been described by Halfon and Mechante [13, 33]. Besides the small size of our cohort that may have been responsible for the lack of association found, other factors may play a more relevant role in fibrogenesis than IR in the setting of HIV/HCV coinfection comparing to those with HCV monoinfection. Low CD4+ cell count and history of AIDS, older age and greater length of HCV infection, HCV genotype 3, alcohol consumption, elevated GPT levels and greater waist circumference [7, 24-26] have been previously associated with increased liver fibrosis in a HIV/HCV coinfection context. Among these, only length of HCV infection was different across fibrosis stage groups. Known risk factors for accelerate fibrosis progression are still unable to explain the wide spectrum of evolution of liver disease in HIV/HCV coinfection. Thus, further studies are required to identify other factors potentially associated with liver fibrosis in this setting. The main limitation of our study was the small size of the population. We were unable to distinguish the temporal relationship given the cross-sectional nature of the study. Alcohol use was self reported and not quantified. Lack of data on other potential confounders, such as lipodystrophy, family history of diabetes mellitus could also affect the interpretation of our findings. Finally, the cohort may not be representative of the HIV/HCV coinfected population since the decision to undergo a TE evaluation was - 14 - made by the caring physician and only the ones who accepted to do an IR screening were included. Conclusions In this cohort, HIV/HCV infection was associated with better metabolic profile than HIV monoinfection, except for IR that was similar in both groups. In HIV/HCV coinfected patients, fibrosis grade was associated with differences in NNRTI use, duration of HCV infection and HCV genotype. Patients with liver fibrosis had higher GOT levels and lower TC, Non-HDL-C and albumin. TC and albumin were independently and inversely associated with LS value. List of abbreviations HCV: Hepatitis C virus; HIV: Human Immunodeficiency virus; cART: Combined antiretroviral therapy; TE: Transient Elastography; LS: Liver stiffness; IR: Insulin resistance; HOMA-IR: Homeostasis model assessment of insulin resistance; QUICKI: Quantitative insulin sensitivity check index; PI: Protease inhibitors; NNRTI: Non-Nucleoside Reverse Transcriptase Inhibitors; NRTI: Nucleoside Reverse Transcriptase Inhibitors; TC: total cholesterol; LDL-C: low density lipoprotein cholesterol; HDL-C: low density lipoprotein cholesterol; TGL: triglycerides; GOT: glutamic oxaloacetic transaminase; GPT: glutamic pyruvic transaminase; GGT: Gamma-glutamyl transpeptidase; ALP: Alkaline phosphatase. Competing interests The authors declare that they have no competing interests. - 15 - Authors' contributions CCB conceived the study, participated in its design, in the acquisition of data and drafted the manuscript; EL conceived the study, participated in its design and revised critically the manuscript; JO participated in its design and revised critically the manuscript; ACS performed the statistical analysis and revised critically the manuscript; LFP performed the statistical analysis; DC and AS revised critically the manuscript. PF conceived the study, participated in its design, in the acquisition of data and revised critically the manuscript. All authors read and approved the final manuscript. Authors' information 1University of Porto Medical School, Alameda Hernani Monteiro, 4200 - 319 Porto, Portugal. 2Endocrinology, Diabetes and Metabolism Department of Centro Hospitalar São João, E.P.E., University of Porto Medical School, Alameda Hernani Monteiro, 4200 - 319 Porto, Portugal. 3Clinical Epidemiology, Predictive Medicine and Public Health Department, University of Porto Medical School, Porto, Portugal and University of Porto Institute of Public Health, Alameda Hernani Monteiro, 4200 - 319 Porto, Portugal. 4 Infeccious Diseases Department of Centro Hospitalar de São João, E.P.E., Alameda Hernani Monteiro, 4200 - 319 Porto, Portugal. Acknowledgements We thank all nurses and physicians who provided support for data collection and extraction. We gratefully acknowledge the patients who volunteered to participate in the study. - 16 - References 1. Operskalski EA, Kovacs A: HIV/HCV co-infection: pathogenesis, clinical complications, treatment, and new therapeutic technologies. 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Petta S, Camma C, Di Marco V, Alessi N, Cabibi D, Caldarella R, Licata A, Massenti F, Tarantino G, Marchesini G et al: Insulin resistance and diabetes increase fibrosis in the liver of patients with genotype 1 HCV infection. The American journal of gastroenterology 2008, 103(5):1136-1144. 39. D'Souza R, Sabin CA, Foster GR: Insulin resistance plays a significant role in liver fibrosis in chronic hepatitis C and in the response to antiviral therapy. The American journal of gastroenterology 2005, 100(7):1509-1515. 19 Tables Table 1 - Sample descriptive statistics and comparison between monoinfected HIV patients and coinfected HIV/HCV patients. HIV (n = 236) HIV/HCV (n = 63) p Gender 1 <0.001 Female 95 (40.3) 7 (11.1) Male 141 (59.7) 56 (88.9) Age years 2 44 (11.48) 43 (6.54) 0.074 Duration of HIV infection years 3 8 (5-10) 13 (9-17) <0.001 CD4 cell count 3 504 (338-716) 366 (285-566) 0.001 <50 HIV RNA copies/mL 1 211 (89.4) 56 (88.9) 0.906 cART cumulative exposure years 3 6 (3-10) 9 (5-12) <0.001 ART 1 PI 136 (57.6) 41 (65.1) 0.285 NNRTI 110 (46.6) 21 (33.3) 0.059 NRTI 232 (98.3) 57 (90.5) 0.007 G Duration of HCV infection years 2 10.6 (5.05) HCV RNA log10 3 6.04 (5.386.61) Genotype 1 1 44 (69.8) 3 13 (20.6) 4 6 (9.5) Alcohol consumption 1 85 (36.0) 27 (42.9) 0.319 1 – number (%); 2 – mean (SD); 3 – median (IQR) 20 Table 2 - Clinical and metabolic features and comparison between monoinfected HIV patients and coinfected HIV/HCV patients. HIV HIV/HCV p hjvh BMI kg/m2 1 25.2 (4.3) 22.9 (2.9) <0.001 Waist circumference cm 1 91.6 (11.3) 85.3 (8.4) <0.001 High blood pressure2 82 (34.7) 5 (7.9) <0.001 Antihypertensive medication2 31 (13.1) 1 (1.6) 0.008 Total cholesterol mg/dL 1 227.5 (56.4) 167.3 (35.8) <0.001 LDL cholesterol mg/dL 1 136.4 (48.5) 97.5 (25.0) <0.001 HDL cholesterol mg/dL 1 47.2 (13.8) 46.5 (15.1) 0.717 Non-HDL Cholesterol mg/dL 3 180.0 (142.0-214.0) 127.0 (104.0-139.0) <0.001 Triglycerides mg/dL 1 266.0 (193.5) 146.3 (71.8) <0.001 Antidyslipidemic therapy 1 114 (48.3) 9 (14.3) <0.001 GOT U/L 3 24.0 (19.3-32.8) 41.0 (29.0-68.0) <0.001 GPT U/L 3 24.0 (18.0-37.0) 56.0 (36.0-95.0) <0.001 GGT U/L 3 40.0 (26.0-76.5) 95.0 (46.0-165.0) <0.001 ALP U/L 3 82.50 (64.0-105.0) 93.0 (76.0-115.0) 0.008 Total bilirubin U/L 3 0.98 (0.96) 0.93 (0.59) 0.068 Albumin g/L 1 44.1 (3.3) 43.0 (4.4) <0.001 Platelets x109/L 1 215.9 (58.1) 172.4 (65.9) 0.039 Prothrombin time mg/dL 1 --- 12.3 (1.4) Fibrinogen mg/dL 1 --- 335.2 (92.1) Liver stiffness kPa 3 --- 7.1 (5.4-9.8) HOMA-IR 3 1.8 (1.2-3.1) 2.0 (1.0-3.3) 0.855 HOMA-IR ≥ 2 2 105 (44.5) 32 (50.8) 0.372 HOMA-IR ≧4 2 37 (15.7) 11 (17.5) 0.732 QUICKI 3 0.35 (0.32-0.37) 0.34 (0.32-0.38) 0.855 Glucose mg/dL 3 90.5 (83.0-98.8) 84.0 (78.0-95.0) 0.001 Insulin mU/L 3 8.1 (5.3-13.1) 9.6 (5.1-14.6) 0.347 HbA1C % 1 5.3 (0.49) 5.1 (0.63) 0.055 1 – mean (SD); 2 – number (%); 3 – median (min, max | IQR) 21 Table 3 - Main features of the HIV/HCV coinfected patients according to liver fibrosis stage. Without Significant Fibrosis Undetermined Fibrosis Significant Fibrosis Cirrhosis p (n=22) (n=21) (n=14) (n=6) Jhh Gender 1 0.700 Female 4 (18.2) 2 (9.5) 1 (7.1) 0 (0.0) Male 18 (81.8) 19 (90.5) 13 (92.9) 6 (100.0) Age 2 44.3 (6.7) 40.1 (6.4) 44.1 (5.8) 42.3 (6.5) 0.163 Duration of HIV infection years 2 13.1 (6.2) 12.3 (5.0) 12.6 (5.8) 12.2 (4.1) 0.959 CD4 cell count 3 444 (297-642) 367 (291-560) 327 (236-507) 346 (143-375) 0.344 <50 HIV RNA copies/mL 1 19 (86.4) 20 (97.2) 12 (85.7) 5 (83.3) 0.887 cART cumulative exposure years 2 9.4 (4.7) 9.2 (4.9) 9.1 (5.2) 6.0 (4.4) 0.492 ART 1 PI 11 (50.0) 16 (76.2) 10 (78.6) 4 (66.7) 0.349 NNRTI 12 (54.5) 6 (28.6) 3 (21.4) 0 (0) 0.040 NRTI 20 (90.2) 19 (90.5) 12 (85.7) 6 (100.0) 0.933 G Duration of HCV infection years 2 11.7 (5.8) 8.6 (4.1) 9.9 (4.5) 15.0 (2.4) 0.022 HCV RNA log10 3 6.3 (5.6-6.9) 5.8 (5.0-6.5) 6.2 (5.5-6.7) 5.7 (4.8-6.3) 0.410 Genotype 1 0,017 1 14 (63.6) 12 (57.1) 12 (85.7) 6 (100.0) 3 3 (13.6) 9 (42.9) 1 (7.1) 0 (0.0) 4 5 (22.5) 0 (0.0) 1 (7.1) 0 (0.0) sjgsh Alcohol consumption1 9 (40.9) 8 (38.1) 7 (50.0) 3 (50.0) 0.887 1 – number (%); 2 – mean (SD); 3 – median (IQR) 11/3/2014 BMC Infectious Diseases | Instructions for Authors | Research articles http://www.biomedcentral.com/bmcinfectdis/authors/instructions/researcharticle 2/6 General guidelines of the journal's style and language are given below. Overview of manuscript sections for Research articles Manuscripts for Research articles submitted to BMC Infectious Diseases should be divided into the following sections (in this order): (if any) (if any) The Accession Numbers of any nucleic acid sequences, protein sequences or atomic coordinates cited in the manuscript should be provided, in square brackets and include the corresponding database name; for example, [EMBL:AB026295, EMBL:AC137000, DDBJ:AE000812, GenBank:U49845, PDB:1BFM, Swiss-Prot:Q96KQ7, PIR:S66116]. The databases for which we can provide direct links are: EMBL Nucleotide Sequence Database (EMBL), DNA Data Bank of Japan (DDBJ), GenBank at the NCBI (GenBank), Protein Data Bank (PDB), Protein Information Resource (PIR) and the Swiss-Prot Protein Database (Swiss-Prot). You can download a template (Mac and Windows compatible; Microsoft Word 98/2000) for your article. For reporting standards please see the information in the About section. Title page The title page should: provide the title of the article list the full names, institutional addresses and email addresses for all authors indicate the corresponding author Please note: the title should include the study design, for example "A versus B in the treatment of C: a randomized controlled trial X is a risk factor for Y: a case control study" abbreviations within the title should be avoided Abstract The Abstract of the manuscript should not exceed 350 words and must be structured into separate sections: Background, the context and purpose of the study; Methods, how the study was performed and statistical tests used; Results, the main findings; Conclusions, brief summary and potential implications. Please minimize the use of abbreviations and do not cite references in the abstract. Trial registration, if your research article reports the results of a controlled health care intervention, please list your trial registry, along with the unique identifying number (e.g. Trial registration: Current Controlled Trials ISRCTN73824458). Please note that there should be no space between the letters and numbers of your trial registration number. We recommend manuscripts that report randomized controlled trials follow the CONSORT extension for abstracts. Keywords Three to ten keywords representing the main content of the article. Background The Background section should be written in a way that is accessible to researchers without specialist knowledge in that area and must clearly state - and, if helpful, illustrate - the background to the research and its aims. Reports of clinical research should, where appropriate, include a summary of a search of the literature to indicate why this study was necessary and what it aimed to contribute to the field. The section should end with a brief statement of what is being reported in the article. Methods The methods section should include the design of the study, the setting, the type of participants or materials involved, a clear description of all interventions and comparisons, and the type of analysis used, including a power calculation if appropriate. Generic drug names should generally be used. When proprietary brands are used in research, include the brand names in parentheses in the Methods section. For studies involving human participants a statement detailing ethical approval and consent should be included in the methods section. For further details of the journal's editorial policies and ethical guidelines see 'About this journal'. For further details of the journal's data-release policy, see the policy section in 'About this journal'. Results and discussion The Results and discussion may be combined into a single section or presented separately. Results of statistical analysis should include, where appropriate, relative and absolute risks or risk reductions, and confidence intervals. The Results and discussion sections may also be broken into subsections with short, informative headings. Conclusions This should state clearly the main conclusions of the research and give a clear explanation of their importance and relevance. Summary illustrations may be included. Title page Abstract Keywords Background Methods Results and discussion Conclusions List of abbreviations used Competing interests Authors' contributions Authors' information Acknowledgements Endnotes References Illustrations and figures Tables and captions Preparing additional files 11/3/2014 BMC Infectious Diseases | Instructions for Authors | Research articles http://www.biomedcentral.com/bmcinfectdis/authors/instructions/researcharticle 3/6 List of abbreviations If abbreviations are used in the text they should be defined in the text at first use, and a list of abbreviations can be provided, which should precede the competing interests and authors' contributions. Competing interests A competing interest exists when your interpretation of data or presentation of information may be influenced by your personal or financial relationship with other people or organizations. Authors must disclose any financial competing interests; they should also reveal any non-financial competing interests that may cause them embarrassment were they to become public after the publication of the manuscript. Authors are required to complete a declaration of competing interests. All competing interests that are declared will be listed at the end of published articles. Where an author gives no competing interests, the listing will read 'The author(s) declare that they have no competing interests'. When completing your declaration, please consider the following questions: Financial competing interests In the past five years have you received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? Is such an organization financing this manuscript (including the article-processing charge)? If so, please specify. Do you hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future? If so, please specify. Do you hold or are you currently applying for any patents relating to the content of the manuscript? Have you received reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript? If so, please specify. Do you have any other financial competing interests? If so, please specify. Non-financial competing interests Are there any non-financial competing interests (political, personal, religious, ideological, academic, intellectual, commercial or any other) to declare in relation to this manuscript? If so, please specify. If you are unsure as to whether you, or one your co-authors, has a competing interest please discuss it with the editorial office. Authors' contributions In order to give appropriate credit to each author of a paper, the individual contributions of authors to the manuscript should be specified in this section. According to ICMJE guidelines, An 'author' is generally considered to be someone who has made substantive intellectual contributions to a published study. To qualify as an author one should 1) have made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; 2) have been involved in drafting the manuscript or revising it critically for important intellectual content; 3) have given final approval of the version to be published; and 4) agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content. Acquisition of funding, collection of data, or general supervision of the research group, alone, does not justify authorship. We suggest the following kind of format (please use initials to refer to each author's contribution): AB carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. JY carried out the immunoassays. MT participated in the sequence alignment. ES participated in the design of the study and performed the statistical analysis. FG conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. All contributors who do not meet the criteria for authorship should be listed in an acknowledgements section. Examples of those who might be acknowledged include a person who provided purely technical help, writing assistance, or a department chair who provided only general support. Authors' information You may choose to use this section to include any relevant information about the author(s) that may aid the reader's interpretation of the article, and understand the standpoint of the author(s). This may include details about the authors' qualifications, current positions they hold at institutions or societies, or any other relevant background information. Please refer to authors using their initials. Note this section should not be used to describe any competing interests. Acknowledgements Please acknowledge anyone who contributed towards the article by making substantial contributions to conception, design, acquisition of data, or analysis and interpretation of data, or who was involved in drafting the manuscript or revising it critically for important intellectual content, but who does not meet the criteria for authorship. Please also include the source(s) of funding for each author, and for the manuscript preparation. Authors must describe the role of the funding body, if any, in design, in the collection, analysis, and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. Please also acknowledge anyone who contributed materials essential for the study. If a language editor has made significant revision of the manuscript, we recommend that you acknowledge the editor by name, where possible. The role of a scientific (medical) writer must be included in the acknowledgements section, including their source(s) of funding. We suggest wording such as 'We thank Jane Doe who provided medical writing services on behalf of XYZ Pharmaceuticals Ltd.' Authors should obtain permission to acknowledge from all those mentioned in the Acknowledgements section. Endnotes Endnotes should be designated within the text using a superscript lowercase letter and all notes (along with their corresponding letter) should be included in the Endnotes section. Please format this section in a paragraph rather than a list. References All references, including URLs, must be numbered consecutively, in square brackets, in the order in which they are cited in the text, followed by any in tables or legends. Each reference must have an individual reference number. Please avoid excessive referencing. If automatic numbering systems are used, the reference numbers must be finalized and the bibliography must be fully formatted before submission. Only articles, datasets, clinical trial registration records and abstracts that have been published or are in press, or are available through public e-print/preprint servers, may be cited; unpublished abstracts, unpublished data and personal communications should not be included in the reference list, but may be included in the text and referred to as "unpublished observations" or "personal communications" giving the names of the involved researchers. Obtaining permission to quote personal communications and unpublished data from the cited colleagues is the responsibility of the author. Footnotes are not allowed, but endnotes are permitted. Journal abbreviations follow Index Medicus/MEDLINE. Citations in the reference list should include all named authors, up to the first 30 before adding 'et al.'.. Any in press articles cited within the references and necessary for the reviewers' assessment of the manuscript should be made available if requested by the editorial office. 11/3/2014 BMC Infectious Diseases | Instructions for Authors | Research articles http://www.biomedcentral.com/bmcinfectdis/authors/instructions/researcharticle 4/6 Style files are available for use with popular bibliographic management software: Examples of the BMC Infectious Diseases reference style are shown below. Please ensure that the reference style is followed precisely; if the references are not in the correct style they may have to be retyped and carefully proofread. All web links and URLs, including links to the authors' own websites, should be given a reference number and included in the reference list rather than within the text of the manuscript. They should be provided in full, including both the title of the site and the URL, in the following format: The Mouse Tumor Biology Database [http://tumor.informatics.jax.org/mtbwi/index.do]. If an author or group of authors can clearly be associated with a web link, such as for weblogs, then they should be included in the reference. Examples of the BMC Infectious Diseases reference style Article within a journal Koonin EV, Altschul SF, Bork P: BRCA1 protein products: functional motifs. Nat Genet 1996, 13:266-267. Article within a journal supplement Orengo CA, Bray JE, Hubbard T, LoConte L, Sillitoe I: Analysis and assessment of ab initio three-dimensional prediction, secondary structure, and contacts prediction. Proteins 1999, 43(Suppl 3):149-170. In press article Kharitonov SA, Barnes PJ: Clinical aspects of exhaled nitric oxide. Eur Respir J, in press. Published abstract Zvaifler NJ, Burger JA, Marinova-Mutafchieva L, Taylor P, Maini RN: Mesenchymal cells, stromal derived factor-1 and rheumatoid arthritis [abstract]. Arthritis Rheum 1999, 42:s250. Article within conference proceedings Jones X: Zeolites and synthetic mechanisms. In Proceedings of the First National Conference on Porous Sieves: 27-30 June 1996; Baltimore. Edited by Smith Y. Stoneham: Butterworth-Heinemann; 1996:16-27. Book chapter, or article within a book Schnepf E: From prey via endosymbiont to plastids: comparative studies in dinoflagellates. In Origins of Plastids. Volume 2. 2nd edition. Edited by Lewin RA. New York: Chapman and Hall; 1993:53-76. Whole issue of journal Ponder B, Johnston S, Chodosh L (Eds): Innovative oncology. In Breast Cancer Res 1998, 10:1-72. Whole conference proceedings Smith Y (Ed): Proceedings of the First National Conference on Porous Sieves: 27-30 June 1996; Baltimore. Stoneham: Butterworth-Heinemann; 1996. Complete book Margulis L: Origin of Eukaryotic Cells. New Haven: Yale University Press; 1970. Monograph or book in a series Hunninghake GW, Gadek JE: The alveolar macrophage. In Cultured Human Cells and Tissues. Edited by Harris TJR. New York: Academic Press; 1995:54-56. [Stoner G (Series Editor): Methods and Perspectives in Cell Biology, vol 1.] Book with institutional author Advisory Committee on Genetic Modification: Annual Report. London; 1999. PhD thesis Kohavi R: Wrappers for performance enhancement and oblivious decision graphs. PhD thesis. Stanford University, Computer Science Department; 1995. Link / URL The Mouse Tumor Biology Database [http://tumor.informatics.jax.org/mtbwi/index.do] Link / URL with author(s) Corpas M: The Crowdfunding Genome Project: a personal genomics community with open source values [http://blogs.biomedcentral.com/bmcblog/2012/07/16/the-crowdfunding-genome-project-a-personal-genomics-community-with-open-source-values/] Dataset with persistent identifier Zheng, L-Y; Guo, X-S; He, B; Sun, L-J; Peng, Y; Dong, S-S; Liu, T-F; Jiang, S; Ramachandran, S; Liu, C-M; Jing, H-C (2011): Genome data from sweet and grain sorghum (Sorghum bicolor). GigaScience. http://dx.doi.org/10.5524/100012. Clinical trial registration record with persistent identifier Mendelow, AD (2006): Surgical Trial in Lobar Intracerebral Haemorrhage. Current Controlled Trials. http://dx.doi.org/10.1186/ISRC TN22153967 Preparing illustrations and figures Illustrations should be provided as separate files, not embedded in the text file. Each figure should include a single illustration and should fit on a single page in portrait format. If a figure consists of separate parts, it is important that a single composite illustration file be submitted which contains all parts of the figure. There is no charge for the use of color figures. Please read our figure preparation guidelines for detailed instructions on maximising the quality of your figures. Formats The following file formats can be accepted: PDF (preferred format for diagrams) DOCX/DOC (single page only) PPTX/PPT (single slide only) EPS BibTeX EndNote style file Reference Manager Zotero 11/3/2014 BMC Infectious Diseases | Instructions for Authors | Research articles http://www.biomedcentral.com/bmcinfectdis/authors/instructions/researcharticle 5/6 PNG (preferred format for photos or images) TIFF JPEG BMP Figure legends The legends should be included in the main manuscript text file at the end of the document, rather than being a part of the figure file. For each figure, the following information should be provided: Figure number (in sequence, using Arabic numerals - i.e. Figure 1, 2, 3 etc); short title of figure (maximum 15 words); detailed legend, up to 300 words. Please note that it is the responsibility of the author(s) to obtain permission from the copyright holder to reproduce figures or tables that have previously been published elsewhere. Preparing tables Each table should be numbered and cited in sequence using Arabic numerals (i.e. Table 1, 2, 3 etc.). Tables should also have a title (above the table) that summarizes the whole table; it should be no longer than 15 words. Detailed legends may then follow, but they should be concise. Tables should always be cited in text in consecutive numerical order. Smaller tables considered to be integral to the manuscript can be pasted into the end of the document text file, in A4 portrait or landscape format. These will be typeset and displayed in the final published form of the article. Such tables should be formatted using the 'Table object' in a word processing program to ensure that columns of data are kept aligned when the file is sent electronically for review; this will not always be the case if columns are generated by simply using tabs to separate text. Columns and rows of data should be made visibly distinct by ensuring that the borders of each cell display as black lines. Commas should not be used to indicate numerical values. Color and shading may not be used; parts of the table can be highlighted using symbols or bold text, the meaning of which should be explained in a table legend. Tables should not be embedded as figures or spreadsheet files. Larger datasets or tables too wide for a portrait page can be uploaded separately as additional files. Additional files will not be displayed in the final, laid-out PDF of the article, but a link will be provided to the files as supplied by the author. Tabular data provided as additional files can be uploaded as an Excel spreadsheet (.xls ) or comma separated values (.csv). As with all files, please use the standard file extensions. Preparing additional files Although BMC Infectious Diseases does not restrict the length and quantity of data included in an article, we encourage authors to provide datasets, tables, movies, or other information as additional files. Please note: All Additional files will be published along with the article. Do not include files such as patient consent forms, certificates of language editing, or revised versions of the main manuscript document with tracked changes. Such files should be sent by email to [email protected], quoting the Manuscript ID number. Results that would otherwise be indicated as "data not shown" can and should be included as additional files. Since many weblinks and URLs rapidly become broken, BMC Infectious Diseases requires that supporting data are included as additional files, or deposited in a recognized repository. Please do not link to data on a personal/departmental website. The maximum file size for additional files is 20 MB each, and files will be virus-scanned on submission. Additional files can be in any format, and will be downloadable from the final published article as supplied by the author. We recommend CSV rather than PDF for tabular data. Certain supported files formats are recognized and can be displayed to the user in the browser. These include most movie formats (for users with the Quicktime plugin), mini-websites prepared according to our guidelines, chemical structure files (MOL, PDB), geographic data files (KML). If additional material is provided, please list the following information in a separate section of the manuscript text: File name (e.g. Additional file 1) File format including the correct file extension for example .pdf, .xls, .txt, .pptx (including name and a URL of an appropriate viewer if format is unusual) Title of data Description of data Additional files should be named "Additional file 1" and so on and should be referenced explicitly by file name within the body of the article, e.g. 'An additional movie file shows this in more detail [see Additional file 1]'. Additional file formats Ideally, file formats for additional files should not be platform-specific, and should be viewable using free or widely available tools. The following are examples of suitable formats. Additional documentation PDF (Adode Acrobat) Animations SWF (Shockwave Flash) Movies MP4 (MPEG 4) MOV (Quicktime) Tabular data XLS, XLSX (Excel Spreadsheet) CSV (Comma separated values) As with figure files, files should be given the standard file extensions. Mini-websites Small self-contained websites can be submitted as additional files, in such a way that they will be browsable from within the full text HTML version of the article. In 11/3/2014 BMC Infectious Diseases | Instructions for Authors | Research articles http://www.biomedcentral.com/bmcinfectdis/authors/instructions/researcharticle 6/6 order to do this, please follow these instructions: Create a folder containing a starting file called index.html (or index.htm) in the root. Put all files necessary for viewing the mini-website within the folder, or sub-folders. Ensure that all links are relative (ie "images/picture.jpg" rather than "/images/picture.jpg" or "http://yourdomain.net/images/picture.jpg" or "C:\Documents and Settings\username\My Documents\mini-website\images\picture.jpg") and no link is longer than 255 characters. Access the index.html file and browse around the mini-website, to ensure that the most commonly used browsers (Internet Explorer and Firefox) are able to view all parts of the mini-website without problems, it is ideal to check this on a different machine. Compress the folder into a ZIP, check the file size is under 20 MB, ensure that index.html is in the root of the ZIP, and that the file has .zip extension, then submit as an additional file with your article. Style and language General Currently, BMC Infectious Diseases can only accept manuscripts written in English. Spelling should be US English or British English, but not a mixture. There is no explicit limit on the length of articles submitted, but authors are encouraged to be concise. BMC Infectious Diseases will not edit submitted manuscripts for style or language; reviewers may advise rejection of a manuscript if it is compromised by grammatical errors. Authors are advised to write clearly and simply, and to have their article checked by colleagues before submission. In-house copyediting will be minimal. Nonnative speakers of English may choose to make use of a copyediting service. Language editing For authors who wish to have the language in their manuscript edited by a native-English speaker with scientific expertise, BioMed Central recommends Edanz. BioMed Central has arranged a 10% discount to the fee charged to BioMed Central authors by Edanz. Use of an editing service is neither a requirement nor a guarantee of acceptance for publication. Please contact Edanz directly to make arrangements for editing, and for pricing and payment details. Help and advice on scientific writing The abstract is one of the most important parts of a manuscript. For guidance, please visit our page on Writing titles and abstracts for scientific articles. Tim Albert has produced for BioMed Central a list of tips for writing a scientific manuscript. American Scientist also provides a list of resources for science writing. For more detailed guidance on preparing a manuscript and writing in English, please visit the BioMed Central author academy. Abbreviations Abbreviations should be used as sparingly as possible. They should be defined when first used and a list of abbreviations can be provided following the main manuscript text. Typography Please use double line spacing. Type the text unjustified, without hyphenating words at line breaks. Use hard returns only to end headings and paragraphs, not to rearrange lines. Capitalize only the first word, and proper nouns, in the title. All lines and pages should be numbered. Authors are asked to ensure that line numbering is included in the main text file of their manuscript at the time of submission to facilitate peer-review. Once a manuscript has been accepted, line numbering should be removed from the manuscript before publication. For authors submitting their manuscript in Microsoft Word please do not insert page breaks in your manuscript to ensure page numbering is consistent between your text file and the PDF generated from your submission and used in the review process. Use the BMC Infectious Diseases . Footnotes are not allowed, but endnotes are permitted. Please do not format the text in multiple columns. Greek and other special characters may be included. If you are unable to reproduce a particular special character, please type out the name of the symbol in full. Please ensure that all special characters used are embedded in the text, otherwise they will be lost during conversion to PDF. Units SI units should be used throughout (liter and molar are permitted, however). reference format