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

2013/2014 Ca a ina Magalhães Cas elo B anco Me abolic abno mali ies and li e s i ness e alua ed by T ansien Elas og aphy in HIV and HIV/HCV coin ec ed pa ien s ma ço, 2014 Mes ado In eg ado em Medicina Á ea: Endoc inologia T abalho e e uado sob a O ien ação de: Dou o a Paula Isabel Ma ques Simões de F ei as T abalho o ganizado de aco do com as no mas da e is a: BMC In ec ious Diseases Ca a ina Magalhães Cas elo B anco Me abolic abno mali ies and li e s i ness e alua ed by T ansien Elas og aphy in HIV and HIV/HCV coin ec ed pa ien s Ma ço, 2014 - 1 - Me abolic abno mali ies and li e s i ness e alua ed by T ansien Elas og aphy in HIV and HIV/HCV coin ec ed pa ien s Ca a ina Cas elo-B anco1§, E a Lau1,2, Joana Oli ei a1,2, Ana C is ina San os1,3, Rosá io Se ão4, Luís Fe ei a-Pin o1, An ónio Sa men o1,4, Da ide Ca alho1,2, Paula F ei as1,2 1Uni e si y o Po o Medical School 2Depa men o Endoc inology, Diabe es and Me abolism o Cen o Hospi ala São João, E.P.E. 3Depa men o Clinical Epidemiology, P edic i e Medicine and Public Heal h , Uni e si y o Po o Medical School, and Uni e si y o Po o Ins i u e o Public Heal h, Po o, Po ugal 4 In ec ious Diseases Depa men o Cen o Hospi ala de São João, E.P.E. §Co esponding au ho Email add esses: CCB: ca a inamcb [email protected] - 2 - Abs ac Backg ound Hepa i is C Vi us (HCV) in ec ion is highly p e alen among HIV in ec ed pa ien s. Li e disease and me abolic complica ions a e he majo conce ns abou hei managemen and he main causes o mo bimo ali y. I is s ill no ully unde s ood how he p esence o coin ec ion a ec s he me abolic p o ile and he na u al his o y o he li e disease. Me hods Re ospec i e coho s udy including 299 adul s unde combined an i e o i al he apy (cART) (236 HIV monoin ec ed and 63 HIV/HCV coin ec ed pa ien s). Insulin Resis ance (IR) was de ined as HOMA-IR≥2. Coin ec ed pa ien s we e di ided in o 4 g oups acco ding o he p esence o ib osis de ined by Li e S i ness (LS): 1) absence o signi ican ib osis, i LS≤6.0 kPa; 2) unde e mined ib osis, i 6.0<LS<9.0 kPa; 3) signi ican ib osis, i 9.0≤LS<14.6 kPa; 4) ci hosis, i LS≥14.6 kPa. Resul s HIV/HCV coin ec ed pa ien s p esen ed lowe body mass index, wais ci cum e ence, hype ension and lipid le els in compa ison o HIV mono-in ec ed pa ien s. Glucose me abolism pa ame e s did no di e acco ding o HCV se os a us and IR was ound in app oxima ely hal o he pa ien s in bo h g oups. In HIV/HCV coin ec ed pa ien s, ib osis g ade was associa ed wi h di e ences in non- nucleoside e e se ansc ip ase inhibi o s (NNRTI) use, du a ion o HCV in ec ion and HCV geno ype. Rega ding me abolic cha ac e is ics, highe glu amic oxaloace ic ansaminase (GOT) le els and lowe o al choles e ol (TC), non-high densi y lipop o ein choles e ol (Non-HDL-C) and albumin we e obse ed in he p esence o li e ib osis. - 3 - NNRTI, TC, GOT(ln), albumin, glucose(ln) and age we e included in he linea eg ession o LS. TC [β=-0.038 (-0.074;-0.001)] and albumin [β=-0.896 (-1.191;- 0.602)] we e ound o be in e sely associa ed wi h LS. Conclusions HIV/HCV coin ec ion was associa ed wi h be e me abolic p o ile han HIV monoin ec ion, excep o insulin esis ance ha was simila in bo h g oups. To al choles e ol and albumin we e independen ly and in e sely associa ed wi h li e s i ness. Keywo ds HIV; HCV; Coin ec ion; Li e s i ness; Li e ib osis; Insulin esis ance; HOMA; Dyslipidemia Backg ound Hepa i is C Vi us in ec ion has an ex emely high p e alence among Human Immunode iciency Vi us (HIV) in ec ed pa ien s, a ec ing 4 o 5 million people wo ldwide [1]. In he se ing o HIV/HCV coin ec ion, apid a es o ib osis p og ession we e obse ed [2] and end-s age li e disease is now he p ima y cause o dea h among pa ien s unde cART [3]. HIV/HCV coin ec ion pa hogenesis is s ill no ully unde s ood. Bo h HIV and HCV in ec ions gi e ise o glucose and lipid diso de s, b inging up o discussion how he p esence o coin ec ion in luences he me abolic s a us. Me abolic changes in HIV/HCV coin ec ed pa ien s ha e been associa ed wi h accele a ed p og ession o li e - 4 - disease, lowe a es o esponse o an i-HCV he apy as well as ca dio and ce eb o ascula complica ions [4, 5]. As a esul , he de elopmen o measu es ha may slow down he p og ession o HCV ela ed li e disease should be a p io i y [3]. Ea lie ecogni ion o hese me abolic changes may be an impo an s ep o allow an imp o ed he apeu ic app oach, bo h o he i al in ec ion and o he me abolic diseases [6]. S aging and moni o ing li e ib osis is a key conce n in he managemen o coin ec ed pa ien , which equi es a ool ha is eliable, ela i ely inexpensi e and accu a e. T ansien Elas og aphy (TE) is a non-in asi e es o assess li e ib osis by measu ing li e s i ness, alida ed o p edic signi ican ib osis and ci hosis in HIV/HCV coin ec ed pa ien s. Besides a oiding he complica ions inhe en o li e biopsy, i also ou g ows he la e in e ms o sampling e o s, in e -obse e a iabili y and accep ance by he pa ien s [7, 8]. The aim o his s udy was o e alua e he p e alence o clinical and me abolic pa ame e s, compa ing HIV monoin ec ed wi h HIV/HCV coin ec ed pa ien s and o iden i y he ac o s associa ed wi h signi ican li e ib osis, assessed by TE in HIV/HCV coin ec ed pa ien s. Me hods Subjec s As pa o a c oss-sec ional s udy, 299 non-ins i u ionalized HIV in ec ed Caucasian adul s unde cART ollowed in he In ec ious Diseases and Endoc inology Ou -pa ien Clinic o Cen o Hospi ala de São João we e included: 236 HIV monoin ec ed and 63 HIV/HCV coin ec ed pa ien s. Only he coin ec ed pa ien s who had a TE e alua ion om 2013 we e included. Pa ien s wi h a p e ious diagnosis o - 5 - diabe es o hepa i is B we e excluded. The s udy p o ocol was app o ed by he Hospi al E hics Commi ee and all pa ien s p o ided in o med consen . Clinical assessmen Fo each pa ien , he ollowing in o ma ion was collec ed using a s anda dized p o ocol: demog aphic da a (age, gende ), du a ion o HIV and HCV in ec ion, du a ion o cART, his o y o hype ension, use o an ihype ensi e o lipid lowe ing d ugs and alcohol use. Weigh , heigh , wais ci cum e ence and es ing blood p essu e we e measu ed as p e iously desc ibed [9]. A enous blood sample was d awn a e a 12-hou o e nigh as . All he samples we e analyzed a he cen al labo a o y o ou hospi al. Plasma glucose, insulin, GOT, glu amic py u ic ansaminase (GPT), gamma-glu amyl anspep idase (GGT), alkaline phospha ase (ALP), albumin, pla ele s, o al choles e ol (TC), high densi y lipop o ein choles e ol (HDL-C) and iglyce ides (TGL) we e de e mined using au oma ic s anda d ou ine enzyma ic me hods. The CD4+ cell coun was de e mined by low cy ome y and plasma RNA-HIV and RNA-HCV we e measu ed by a quan i a i e e e se ansc ip ase polyme ase chain eac ion (PCR), which had a lowe limi o de ec ion o 50 copies/mL. HCV in ec ion was diagnosed by he p esence o se um an ibodies agains HCV (RIBA es ) and de ec able se um HCV RNA PCR. Insulin esis ance Insulin esis ance was de ined by he homeos asis model assessmen o insulin esis ance (HOMA-IR) and insulin sensi i i y by he quan i a i e insulin sensi i i y check index (QUICKI). These indexes we e calcula ed by he ollowing o mulas: HOMA-IR index = ( as ing plasma insulin [mU/L] x as ing plasma glucose [mg/dL])/450 [10]. QUICKI = 1/[log ( as ing insulin [mU/L]) +log ( as ing plasma glucose [mg/dL]) - 12 - immune esponse bo h in HIV and HCV in ec ions gi e ise o glucose and lipid me abolic diso de s ha , in u n, a e isk ac o s o li e damage [1, 28]. cART bene i s in educing li e disease p og ession by imp o ing hos immuni y seem o ou weigh i s hepa o oxici y and IR isk [3, 29, 30]. In HCV in ec ion, low lipid le els ha e been co ela ed no only wi h s ea osis and mo e ad anced li e ib osis, bu also wi h non- esponse o in e e on ea men [4]. This is in line wi h wha was obse ed in his s udy: hose wi h highe g ades o ib osis had lowe TC and non-HDL-C. TC emained signi ican ly in e sely associa ed wi h LS, independen ly o age, NNRTI, TC, GOT and albumin. GOT ele a ions a e common wi h ib osis p og ession bu hey a e no a good ma ke , especially in hose wi h HIV in ec ion, in whom he use o cART also con ibu es o al e a ions in he hepa ic p o ile. No mal le el o GOT should no exclude he need o an i-HCV ea men because he e a e pa ien s who de elop ib osis in he absence o ansaminases ele a ion [25]. Lowe albumin is an es ablished ma ke o hepa ic insu iciency. In ou coho , albumin le els we e nega i ely associa ed wi h LS in ou model o li e ib osis, independen ly o he o he a iables included in he mul i a ia ed model. Cu en li e a u e is no ye acco dan ega ding IR impo ance o he pa hogenesis o accele a ed ib osis p og ession in HIV/HCV in ec ed indi iduals, as some s udies desc ibed a posi i e co ela ion be ween IR and mo e ad anced g ades o ib osis [12, 31, 32] and o he s did no ind ha associa ion [13, 33]. Mo eo e , i was epo ed ha IR is associa ed wi h a poo sus ained i ological esponse o HCV ea men in HIV/HCV coin ec ed as well as in HCV monoin ec ed pa ien s [34, 35]. On he o he hand, his ela ionship has been consis en ly epo ed o HCV monoin ec ion, sugges ing ha IR is a de e minan o ib ogenesis in ch onic hepa i is - 13 - C, independen o hepa ic s ea osis, h ough di ec s imula ion o hepa ic s ella e cells by hype glycaemia and hype insulinemia o p oli e a e and o sec e e ex acellula ma ix [36-39]. Concomi an ly, IR p omo es hepa ic lipogenesis and i is plausible ha s ea osis pe se con ibu es o ib ogenesis. No associa ion was ound o glucose (ln) and LS in he mul i a ia ed model, simila ly o wha has been desc ibed by Hal on and Mechan e [13, 33]. Besides he small size o ou coho ha may ha e been esponsible o he lack o associa ion ound, o he ac o s may play a mo e ele an ole in ib ogenesis han IR in he se ing o HIV/HCV coin ec ion compa ing o hose wi h HCV monoin ec ion. Low CD4+ cell coun and his o y o AIDS, olde age and g ea e leng h o HCV in ec ion, HCV geno ype 3, alcohol consump ion, ele a ed GPT le els and g ea e wais ci cum e ence [7, 24-26] ha e been p e iously associa ed wi h inc eased li e ib osis in a HIV/HCV coin ec ion con ex . Among hese, only leng h o HCV in ec ion was di e en ac oss ib osis s age g oups. Known isk ac o s o accele a e ib osis p og ession a e s ill unable o explain he wide spec um o e olu ion o li e disease in HIV/HCV coin ec ion. Thus, u he s udies a e equi ed o iden i y o he ac o s po en ially associa ed wi h li e ib osis in his se ing. The main limi a ion o ou s udy was he small size o he popula ion. We we e unable o dis inguish he empo al ela ionship gi en he c oss-sec ional na u e o he s udy. Alcohol use was sel epo ed and no quan i ied. Lack o da a on o he po en ial con ounde s, such as lipodys ophy, amily his o y o diabe es melli us could also a ec he in e p e a ion o ou indings. Finally, he coho may no be ep esen a i e o he HIV/HCV coin ec ed popula ion since he decision o unde go a TE e alua ion was - 14 - made by he ca ing physician and only he ones who accep ed o do an IR sc eening we e included. Conclusions In his coho , HIV/HCV in ec ion was associa ed wi h be e me abolic p o ile han HIV monoin ec ion, excep o IR ha was simila in bo h g oups. In HIV/HCV coin ec ed pa ien s, ib osis g ade was associa ed wi h di e ences in NNRTI use, du a ion o HCV in ec ion and HCV geno ype. Pa ien s wi h li e ib osis had highe GOT le els and lowe TC, Non-HDL-C and albumin. TC and albumin we e independen ly and in e sely associa ed wi h LS alue. Lis o abb e ia ions HCV: Hepa i is C i us; HIV: Human Immunode iciency i us; cART: Combined an i e o i al he apy; TE: T ansien Elas og aphy; LS: Li e s i ness; IR: Insulin esis ance; HOMA-IR: Homeos asis model assessmen o insulin esis ance; QUICKI: Quan i a i e insulin sensi i i y check index; PI: P o ease inhibi o s; NNRTI: Non-Nucleoside Re e se T ansc ip ase Inhibi o s; NRTI: Nucleoside Re e se T ansc ip ase Inhibi o s; TC: o al choles e ol; LDL-C: low densi y lipop o ein choles e ol; HDL-C: low densi y lipop o ein choles e ol; TGL: iglyce ides; GOT: glu amic oxaloace ic ansaminase; GPT: glu amic py u ic ansaminase; GGT: Gamma-glu amyl anspep idase; ALP: Alkaline phospha ase. Compe ing in e es s The au ho s decla e ha hey ha e no compe ing in e es s. - 15 - Au ho s' con ibu ions CCB concei ed he s udy, pa icipa ed in i s design, in he acquisi ion o da a and d a ed he manusc ip ; EL concei ed he s udy, pa icipa ed in i s design and e ised c i ically he manusc ip ; JO pa icipa ed in i s design and e ised c i ically he manusc ip ; ACS pe o med he s a is ical analysis and e ised c i ically he manusc ip ; LFP pe o med he s a is ical analysis; DC and AS e ised c i ically he manusc ip . PF concei ed he s udy, pa icipa ed in i s design, in he acquisi ion o da a and e ised c i ically he manusc ip . All au ho s ead and app o ed he inal manusc ip . 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Ba ei o P, Ma in-Ca bone o L, Nunez M, Ri as P, Mo en e A, Sima o N, Laba ga P, Gonzalez-Lahoz J, So iano V: P edic o s o li e ib osis in HIV-in ec ed pa ien s wi h ch onic hepa i is C i us (HCV) in ec ion: assessmen using ansien elas ome y and he ole o HCV geno ype 3. Clinical in ec ious diseases : an o icial publica ion o he In ec ious Diseases Socie y o Ame ica 2006, 42(7):1032-1039. 26. Ma in-Ca bone o L, Benhamou Y, Puo i M, Be engue J, Mallolas J, Que eda C, A izco e a A, Gonzalez A, Rocks oh J, Asensi V e al: Incidence and p edic o s o se e e li e ib osis in human immunode iciency i us-in ec ed pa ien s wi h ch onic hepa i is C: a Eu opean collabo a i e s udy. Clinical in ec ious diseases : an o icial publica ion o he In ec ious Diseases Socie y o Ame ica 2004, 38(1):128-133. 27. Lin W, Weinbe g EM, Chung RT: Pa hogenesis o accele a ed ib osis in HIV/HCV co- in ec ion. Jou nal o In ec ious Diseases 2013, 207(suppl 1):S13-S18. 28. Slama L, Le Camus C, Se a y L, Pialoux G, Capeau J, Gha akhanian S: Me abolic diso de s and ch onic i al disease: he case o HIV and HCV. Diabe es & me abolism 2009, 35(1):1-11. 18 29. Qu ishi N, K euzbe g C, Luch e s G, E enbe ge W, Kup e B, Saue b uch T, Rocks oh JK, Spengle U: E ec o an i e o i al he apy on li e - ela ed mo ali y in pa ien s wi h HIV and hepa i is C i us coin ec ion. Lance 2003, 362(9397):1708-1713. 30. Loko MA, Bani-Sad F, Winnock M, Lacombe K, Ca ie i P, Neau D, Mo la P, Se a y L, Dabis F, Salmon D: Impac o HAART exposu e and associa ed lipodys ophy on ad anced li e ib osis in HIV/HCV-coin ec ed pa ien s. Jou nal o i al hepa i is 2011, 18(7):e307-314. 31. Me chan e N, Ri e o A, de Los San os-Gil I, Me ino D, Ma quez M, Lopez-Ruz MA, Rod iguez-Bano J, Del Valle J, Camacho A, Sanz-Sanz J e al: Insulin esis ance is associa ed wi h li e s i ness in HIV/HCV co-in ec ed pa ien s. Gu 2009, 58(12):1654-1660. 32. B uno R, Sacchi P, Cima S, Maiocchi L, Pa uno SF, Kle sy C, Ba ba ini G, Zucca o V, Camma C, Filice G: Co ela ion be ween FIB4, li e s i ness and me abolic pa ame e s in pa ien s wi h HIV and hepa i is C i us co-in ec ion. Diges i e and li e disease : o icial jou nal o he I alian Socie y o Gas oen e ology and he I alian Associa ion o he S udy o he Li e 2011, 43(7):575-578. 33. Me chan e N, Macias J, Ramayo E, Ve ga a S, Ga cia-Ga cia JA, Mi a JA, Co zo JE, Gomez-Ma eos JM, Lozano F, Pineda JA: Insulin esis ance is no associa ed wi h li e ib osis p og ession in HIV/hepa i is C i us-coin ec ed pa ien s. Jou nal o i al hepa i is 2006, 13(7):449-456. 34. Nas a P, Ga i F, Puo i M, Cologni G, Be gamaschi V, Bo ghi F, Ma i A, Ricci A, Ca osi G: Insulin esis ance impai s apid i ologic esponse in HIV/hepa i is C i us coin ec ed pa ien s on pegin e e on-al a-2a. AIDS (London, England) 2008, 22(7):857-861. 35. Eslam M, Lopez-Co es LF, Rome o-Gomez M: The ole o insulin esis ance in HIV/hepa i is C i us-coin ec ed pa ien s. Cu en opinion in HIV and AIDS 2011, 6(6):553-558. 36. Pe a S, Camma C, Ma co VD, Macaluso FS, Maida M, Pizzolan i G, Belmon e B, Cabibi D, S e ano RD, Fe a o D: Hepa ic s ea osis and insulin esis ance a e associa ed wi h se e e ib osis in pa ien s wi h ch onic hepa i is caused by HBV o HCV in ec ion. Li e In e na ional 2011, 31(4):507-515. 37. Fa oux L, Poujol-Robe A, Guecho J, Wendum D, Poupon R, Se a y L: Insulin esis ance is a cause o s ea osis and ib osis p og ession in ch onic hepa i is C. Gu 2005, 54(7):1003-1008. 38. Pe a S, Camma C, Di Ma co V, Alessi N, Cabibi D, Calda ella R, Lica a A, Massen i F, Ta an ino G, Ma chesini G e al: Insulin esis ance and diabe es inc ease ib osis in he li e o pa ien s wi h geno ype 1 HCV in ec ion. The Ame ican jou nal o gas oen e ology 2008, 103(5):1136-1144. 39. D'Souza R, Sabin CA, Fos e GR: Insulin esis ance plays a signi ican ole in li e ib osis in ch onic hepa i is C and in he esponse o an i i al he apy. The Ame ican jou nal o gas oen e ology 2005, 100(7):1509-1515. 19 Tables Table 1 - Sample desc ip i e s a is ics and compa ison be ween monoin ec ed HIV pa ien s and coin ec ed HIV/HCV pa ien s. HIV (n = 236) HIV/HCV (n = 63) p Gende 1 <0.001 Female 95 (40.3) 7 (11.1) Male 141 (59.7) 56 (88.9) Age yea s 2 44 (11.48) 43 (6.54) 0.074 Du a ion o HIV in ec ion yea s 3 8 (5-10) 13 (9-17) <0.001 CD4 cell coun 3 504 (338-716) 366 (285-566) 0.001 <50 HIV RNA copies/mL 1 211 (89.4) 56 (88.9) 0.906 cART cumula i e exposu e yea s 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 Du a ion o HCV in ec ion yea s 2 10.6 (5.05) HCV RNA log10 3 6.04 (5.38- 6.61) Geno ype 1 1 44 (69.8) 3 13 (20.6) 4 6 (9.5) Alcohol consump ion 1 85 (36.0) 27 (42.9) 0.319 1 – numbe (%); 2 – mean (SD); 3 – median (IQR) 20 Table 2 - Clinical and me abolic ea u es and compa ison be ween monoin ec ed HIV pa ien s and coin ec ed HIV/HCV pa ien s. HIV HIV/HCV p hj h BMI kg/m2 1 25.2 (4.3) 22.9 (2.9) <0.001 Wais ci cum e ence cm 1 91.6 (11.3) 85.3 (8.4) <0.001 High blood p essu e2 82 (34.7) 5 (7.9) <0.001 An ihype ensi e medica ion2 31 (13.1) 1 (1.6) 0.008 To al choles e ol mg/dL 1 227.5 (56.4) 167.3 (35.8) <0.001 LDL choles e ol mg/dL 1 136.4 (48.5) 97.5 (25.0) <0.001 HDL choles e ol mg/dL 1 47.2 (13.8) 46.5 (15.1) 0.717 Non-HDL Choles e ol mg/dL 3 180.0 (142.0-214.0) 127.0 (104.0-139.0) <0.001 T iglyce ides mg/dL 1 266.0 (193.5) 146.3 (71.8) <0.001 An idyslipidemic he apy 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 To al bili ubin 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 Pla ele s x109/L 1 215.9 (58.1) 172.4 (65.9) 0.039 P o h ombin ime mg/dL 1 --- 12.3 (1.4) Fib inogen mg/dL 1 --- 335.2 (92.1) Li e s i ness 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 – numbe (%); 3 – median (min, max | IQR) 21 Table 3 - Main ea u es o he HIV/HCV coin ec ed pa ien s acco ding o li e ib osis s age. Wi hou Signi ican Fib osis Unde e mined Fib osis Signi ican Fib osis Ci hosis p (n=22) (n=21) (n=14) (n=6) Jhh Gende 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 Du a ion o HIV in ec ion yea s 2 13.1 (6.2) 12.3 (5.0) 12.6 (5.8) 12.2 (4.1) 0.959 CD4 cell coun 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 cumula i e exposu e yea s 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 Du a ion o HCV in ec ion yea s 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 Geno ype 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 consump ion1 9 (40.9) 8 (38.1) 7 (50.0) 3 (50.0) 0.887 1 – numbe (%); 2 – mean (SD); 3 – median (IQR) 11/3/2014 BMC In ec ious Diseases | Ins uc ions o Au ho s | Resea ch a icles h p://www.biomedcen al.com/bmcin ec dis/au ho s/ins uc ions/ esea cha icle 2/6 Gene al guidelines o he jou nal's s yle and language a e gi en below. O e iew o manusc ip sec ions o Resea ch a icles Manusc ip s o Resea ch a icles submi ed o BMC In ec ious Diseases should be di ided in o he ollowing sec ions (in his o de ): (i any) (i any) The Accession Numbe s o any nucleic acid sequences, p o ein sequences o a omic coo dina es ci ed in he manusc ip should be p o ided, in squa e b acke s and include he co esponding da abase name; o example, [EMBL:AB026295, EMBL:AC137000, DDBJ:AE000812, GenBank:U49845, PDB:1BFM, Swiss-P o :Q96KQ7, PIR:S66116]. The da abases o which we can p o ide di ec links a e: EMBL Nucleo ide Sequence Da abase (EMBL), DNA Da a Bank o Japan (DDBJ), GenBank a he NCBI (GenBank), P o ein Da a Bank (PDB), P o ein In o ma ion Resou ce (PIR) and he Swiss-P o P o ein Da abase (Swiss-P o ). You can download a empla e (Mac and Windows compa ible; Mic oso Wo d 98/2000) o you a icle. Fo epo ing s anda ds please see he in o ma ion in he Abou sec ion. Ti le page The i le page should: p o ide he i le o he a icle lis he ull names, ins i u ional add esses and email add esses o all au ho s indica e he co esponding au ho Please no e: he i le should include he s udy design, o example "A e sus B in he ea men o C: a andomized con olled ial X is a isk ac o o Y: a case con ol s udy" abb e ia ions wi hin he i le should be a oided Abs ac The Abs ac o he manusc ip should no exceed 350 wo ds and mus be s uc u ed in o sepa a e sec ions: Backg ound, he con ex and pu pose o he s udy; Me hods, how he s udy was pe o med and s a is ical es s used; Resul s, he main indings; Conclusions, b ie summa y and po en ial implica ions. Please minimize he use o abb e ia ions and do no ci e e e ences in he abs ac . T ial egis a ion, i you esea ch a icle epo s he esul s o a con olled heal h ca e in e en ion, please lis you ial egis y, along wi h he unique iden i ying numbe (e.g. T ial egis a ion: Cu en Con olled T ials ISRCTN73824458). Please no e ha he e should be no space be ween he le e s and numbe s o you ial egis a ion numbe . We ecommend manusc ip s ha epo andomized con olled ials ollow he CONSORT ex ension o abs ac s. Keywo ds Th ee o en keywo ds ep esen ing he main con en o he a icle. Backg ound The Backg ound sec ion should be w i en in a way ha is accessible o esea che s wi hou specialis knowledge in ha a ea and mus clea ly s a e - and, i help ul, illus a e - he backg ound o he esea ch and i s aims. Repo s o clinical esea ch should, whe e app op ia e, include a summa y o a sea ch o he li e a u e o indica e why his s udy was necessa y and wha i aimed o con ibu e o he ield. The sec ion should end wi h a b ie s a emen o wha is being epo ed in he a icle. Me hods The me hods sec ion should include he design o he s udy, he se ing, he ype o pa icipan s o ma e ials in ol ed, a clea desc ip ion o all in e en ions and compa isons, and he ype o analysis used, including a powe calcula ion i app op ia e. Gene ic d ug names should gene ally be used. When p op ie a y b ands a e used in esea ch, include he b and names in pa en heses in he Me hods sec ion. Fo s udies in ol ing human pa icipan s a s a emen de ailing e hical app o al and consen should be included in he me hods sec ion. Fo u he de ails o he jou nal's edi o ial policies and e hical guidelines see 'Abou his jou nal'. Fo u he de ails o he jou nal's da a- elease policy, see he policy sec ion in 'Abou his jou nal'. Resul s and discussion The Resul s and discussion may be combined in o a single sec ion o p esen ed sepa a ely. Resul s o s a is ical analysis should include, whe e app op ia e, ela i e and absolu e isks o isk educ ions, and con idence in e als. The Resul s and discussion sec ions may also be b oken in o subsec ions wi h sho , in o ma i e headings. Conclusions This should s a e clea ly he main conclusions o he esea ch and gi e a clea explana ion o hei impo ance and ele ance. Summa y illus a ions may be included. Ti le page Abs ac Keywo ds Backg ound Me hods Resul s and discussion Conclusions Lis o abb e ia ions used Compe ing in e es s Au ho s' con ibu ions Au ho s' in o ma ion Acknowledgemen s Endno es Re e ences Illus a ions and igu es Tables and cap ions P epa ing addi ional iles 11/3/2014 BMC In ec ious Diseases | Ins uc ions o Au ho s | Resea ch a icles h p://www.biomedcen al.com/bmcin ec dis/au ho s/ins uc ions/ esea cha icle 3/6 Lis o abb e ia ions I abb e ia ions a e used in he ex hey should be de ined in he ex a i s use, and a lis o abb e ia ions can be p o ided, which should p ecede he compe ing in e es s and au ho s' con ibu ions. Compe ing in e es s A compe ing in e es exis s when you in e p e a ion o da a o p esen a ion o in o ma ion may be in luenced by you pe sonal o inancial ela ionship wi h o he people o o ganiza ions. Au ho s mus disclose any inancial compe ing in e es s; hey should also e eal any non- inancial compe ing in e es s ha may cause hem emba assmen we e hey o become public a e he publica ion o he manusc ip . Au ho s a e equi ed o comple e a decla a ion o compe ing in e es s. All compe ing in e es s ha a e decla ed will be lis ed a he end o published a icles. Whe e an au ho gi es no compe ing in e es s, he lis ing will ead 'The au ho (s) decla e ha hey ha e no compe ing in e es s'. When comple ing you decla a ion, please conside he ollowing ques ions: Financial compe ing in e es s In he pas i e yea s ha e you ecei ed eimbu semen s, ees, unding, o sala y om an o ganiza ion ha may in any way gain o lose inancially om he publica ion o his manusc ip , ei he now o in he u u e? Is such an o ganiza ion inancing his manusc ip (including he a icle-p ocessing cha ge)? I so, please speci y. Do you hold any s ocks o sha es in an o ganiza ion ha may in any way gain o lose inancially om he publica ion o his manusc ip , ei he now o in he u u e? I so, please speci y. Do you hold o a e you cu en ly applying o any pa en s ela ing o he con en o he manusc ip ? Ha e you ecei ed eimbu semen s, ees, unding, o sala y om an o ganiza ion ha holds o has applied o pa en s ela ing o he con en o he manusc ip ? I so, please speci y. Do you ha e any o he inancial compe ing in e es s? I so, please speci y. Non- inancial compe ing in e es s A e he e any non- inancial compe ing in e es s (poli ical, pe sonal, eligious, ideological, academic, in ellec ual, comme cial o any o he ) o decla e in ela ion o his manusc ip ? I so, please speci y. I you a e unsu e as o whe he you, o one you co-au ho s, has a compe ing in e es please discuss i wi h he edi o ial o ice. Au ho s' con ibu ions In o de o gi e app op ia e c edi o each au ho o a pape , he indi idual con ibu ions o au ho s o he manusc ip should be speci ied in his sec ion. Acco ding o ICMJE guidelines, An 'au ho ' is gene ally conside ed o be someone who has made subs an i e in ellec ual con ibu ions o a published s udy. To quali y as an au ho one should 1) ha e made subs an ial con ibu ions o concep ion and design, o acquisi ion o da a, o analysis and in e p e a ion o da a; 2) ha e been in ol ed in d a ing he manusc ip o e ising i c i ically o impo an in ellec ual con en ; 3) ha e gi en inal app o al o he e sion o be published; and 4) ag ee o be accoun able o all aspec s o he wo k in ensu ing ha ques ions ela ed o he accu acy o in eg i y o any pa o he wo k a e app op ia ely in es iga ed and esol ed. Each au ho should ha e pa icipa ed su icien ly in he wo k o ake public esponsibili y o app op ia e po ions o he con en . Acquisi ion o unding, collec ion o da a, o gene al supe ision o he esea ch g oup, alone, does no jus i y au ho ship. We sugges he ollowing kind o o ma (please use ini ials o e e o each au ho 's con ibu ion): AB ca ied ou he molecula gene ic s udies, pa icipa ed in he sequence alignmen and d a ed he manusc ip . JY ca ied ou he immunoassays. MT pa icipa ed in he sequence alignmen . ES pa icipa ed in he design o he s udy and pe o med he s a is ical analysis. FG concei ed o he s udy, and pa icipa ed in i s design and coo dina ion and helped o d a he manusc ip . All au ho s ead and app o ed he inal manusc ip . All con ibu o s who do no mee he c i e ia o au ho ship should be lis ed in an acknowledgemen s sec ion. Examples o hose who migh be acknowledged include a pe son who p o ided pu ely echnical help, w i ing assis ance, o a depa men chai who p o ided only gene al suppo . Au ho s' in o ma ion You may choose o use his sec ion o include any ele an in o ma ion abou he au ho (s) ha may aid he eade 's in e p e a ion o he a icle, and unde s and he s andpoin o he au ho (s). This may include de ails abou he au ho s' quali ica ions, cu en posi ions hey hold a ins i u ions o socie ies, o any o he ele an backg ound in o ma ion. Please e e o au ho s using hei ini ials. No e his sec ion should no be used o desc ibe any compe ing in e es s. Acknowledgemen s Please acknowledge anyone who con ibu ed owa ds he a icle by making subs an ial con ibu ions o concep ion, design, acquisi ion o da a, o analysis and in e p e a ion o da a, o who was in ol ed in d a ing he manusc ip o e ising i c i ically o impo an in ellec ual con en , bu who does no mee he c i e ia o au ho ship. Please also include he sou ce(s) o unding o each au ho , and o he manusc ip p epa a ion. Au ho s mus desc ibe he ole o he unding body, i any, in design, in he collec ion, analysis, and in e p e a ion o da a; in he w i ing o he manusc ip ; and in he decision o submi he manusc ip o publica ion. Please also acknowledge anyone who con ibu ed ma e ials essen ial o he s udy. I a language edi o has made signi ican e ision o he manusc ip , we ecommend ha you acknowledge he edi o by name, whe e possible. The ole o a scien i ic (medical) w i e mus be included in he acknowledgemen s sec ion, including hei sou ce(s) o unding. We sugges wo ding such as 'We hank Jane Doe who p o ided medical w i ing se ices on behal o XYZ Pha maceu icals L d.' Au ho s should ob ain pe mission o acknowledge om all hose men ioned in he Acknowledgemen s sec ion. Endno es Endno es should be designa ed wi hin he ex using a supe sc ip lowe case le e and all no es (along wi h hei co esponding le e ) should be included in he Endno es sec ion. Please o ma his sec ion in a pa ag aph a he han a lis . Re e ences All e e ences, including URLs, mus be numbe ed consecu i ely, in squa e b acke s, in he o de in which hey a e ci ed in he ex , ollowed by any in ables o legends. Each e e ence mus ha e an indi idual e e ence numbe . Please a oid excessi e e e encing. I au oma ic numbe ing sys ems a e used, he e e ence numbe s mus be inalized and he bibliog aphy mus be ully o ma ed be o e submission. Only a icles, da ase s, clinical ial egis a ion eco ds and abs ac s ha ha e been published o a e in p ess, o a e a ailable h ough public e-p in /p ep in se e s, may be ci ed; unpublished abs ac s, unpublished da a and pe sonal communica ions should no be included in he e e ence lis , bu may be included in he ex and e e ed o as "unpublished obse a ions" o "pe sonal communica ions" gi ing he names o he in ol ed esea che s. Ob aining pe mission o quo e pe sonal communica ions and unpublished da a om he ci ed colleagues is he esponsibili y o he au ho . Foo no es a e no allowed, bu endno es a e pe mi ed. Jou nal abb e ia ions ollow Index Medicus/MEDLINE. Ci a ions in he e e ence lis should include all named au ho s, up o he i s 30 be o e adding 'e al.'.. Any in p ess a icles ci ed wi hin he e e ences and necessa y o he e iewe s' assessmen o he manusc ip should be made a ailable i eques ed by he edi o ial o ice. 11/3/2014 BMC In ec ious Diseases | Ins uc ions o Au ho s | Resea ch a icles h p://www.biomedcen al.com/bmcin ec dis/au ho s/ins uc ions/ esea cha icle 4/6 S yle iles a e a ailable o use wi h popula bibliog aphic managemen so wa e: Examples o he BMC In ec ious Diseases e e ence s yle a e shown below. Please ensu e ha he e e ence s yle is ollowed p ecisely; i he e e ences a e no in he co ec s yle hey may ha e o be e yped and ca e ully p oo ead. All web links and URLs, including links o he au ho s' own websi es, should be gi en a e e ence numbe and included in he e e ence lis a he han wi hin he ex o he manusc ip . They should be p o ided in ull, including bo h he i le o he si e and he URL, in he ollowing o ma : The Mouse Tumo Biology Da abase [h p:// umo .in o ma ics.jax.o g/m bwi/index.do]. I an au ho o g oup o au ho s can clea ly be associa ed wi h a web link, such as o weblogs, hen hey should be included in he e e ence. Examples o he BMC In ec ious Diseases e e ence s yle A icle wi hin a jou nal Koonin EV, Al schul SF, Bo k P: BRCA1 p o ein p oduc s: unc ional mo i s. Na Gene 1996, 13:266-267. A icle wi hin a jou nal supplemen O engo CA, B ay JE, Hubba d T, LoCon e L, Silli oe I: Analysis and assessmen o ab ini io h ee-dimensional p edic ion, seconda y s uc u e, and con ac s p edic ion. P o eins 1999, 43(Suppl 3):149-170. In p ess a icle Kha i ono SA, Ba nes PJ: Clinical aspec s o exhaled ni ic oxide. Eu Respi J, in p ess. Published abs ac Z ai le NJ, Bu ge JA, Ma ino a-Mu a chie a L, Taylo P, Maini RN: Mesenchymal cells, s omal de i ed ac o -1 and heuma oid a h i is [abs ac ]. A h i is Rheum 1999, 42:s250. A icle wi hin con e ence p oceedings Jones X: Zeoli es and syn he ic mechanisms. In P oceedings o he Fi s Na ional Con e ence on Po ous Sie es: 27-30 June 1996; Bal imo e. Edi ed by Smi h Y. S oneham: Bu e wo h-Heinemann; 1996:16-27. Book chap e , o a icle wi hin a book Schnep E: F om p ey ia endosymbion o plas ids: compa a i e s udies in dino lagella es. In O igins o Plas ids. Volume 2. 2nd edi ion. Edi ed by Lewin RA. New Yo k: Chapman and Hall; 1993:53-76. Whole issue o jou nal Ponde B, Johns on S, Chodosh L (Eds): Inno a i e oncology. In B eas Cance Res 1998, 10:1-72. Whole con e ence p oceedings Smi h Y (Ed): P oceedings o he Fi s Na ional Con e ence on Po ous Sie es: 27-30 June 1996; Bal imo e. S oneham: Bu e wo h-Heinemann; 1996. Comple e book Ma gulis L: O igin o Euka yo ic Cells. New Ha en: Yale Uni e si y P ess; 1970. Monog aph o book in a se ies Hunninghake GW, Gadek JE: The al eola mac ophage. In Cul u ed Human Cells and Tissues. Edi ed by Ha is TJR. New Yo k: Academic P ess; 1995:54-56. [S one G (Se ies Edi o ): Me hods and Pe spec i es in Cell Biology, ol 1.] Book wi h ins i u ional au ho Ad iso y Commi ee on Gene ic Modi ica ion: Annual Repo . London; 1999. PhD hesis Koha i R: W appe s o pe o mance enhancemen and obli ious decision g aphs. PhD hesis. S an o d Uni e si y, Compu e Science Depa men ; 1995. Link / URL The Mouse Tumo Biology Da abase [h p:// umo .in o ma ics.jax.o g/m bwi/index.do] Link / URL wi h au ho (s) Co pas M: The C owd unding Genome P ojec : a pe sonal genomics communi y wi h open sou ce alues [h p://blogs.biomedcen al.com/bmcblog/2012/07/16/ he-c owd unding-genome-p ojec -a-pe sonal-genomics-communi y-wi h-open-sou ce- alues/] Da ase wi h pe sis en iden i ie Zheng, L-Y; Guo, X-S; He, B; Sun, L-J; Peng, Y; Dong, S-S; Liu, T-F; Jiang, S; Ramachand an, S; Liu, C-M; Jing, H-C (2011): Genome da a om swee and g ain so ghum (So ghum bicolo ). GigaScience. h p://dx.doi.o g/10.5524/100012. Clinical ial egis a ion eco d wi h pe sis en iden i ie Mendelow, AD (2006): Su gical T ial in Loba In ace eb al Haemo hage. Cu en Con olled T ials. h p://dx.doi.o g/10.1186/ISRC TN22153967 P epa ing illus a ions and igu es Illus a ions should be p o ided as sepa a e iles, no embedded in he ex ile. Each igu e should include a single illus a ion and should i on a single page in po ai o ma . I a igu e consis s o sepa a e pa s, i is impo an ha a single composi e illus a ion ile be submi ed which con ains all pa s o he igu e. The e is no cha ge o he use o colo igu es. Please ead ou igu e p epa a ion guidelines o de ailed ins uc ions on maximising he quali y o you igu es. Fo ma s The ollowing ile o ma s can be accep ed: PDF (p e e ed o ma o diag ams) DOCX/DOC (single page only) PPTX/PPT (single slide only) EPS BibTeX EndNo e s yle ile Re e ence Manage Zo e o 11/3/2014 BMC In ec ious Diseases | Ins uc ions o Au ho s | Resea ch a icles h p://www.biomedcen al.com/bmcin ec dis/au ho s/ins uc ions/ esea cha icle 5/6 PNG (p e e ed o ma o pho os o images) TIFF JPEG BMP Figu e legends The legends should be included in he main manusc ip ex ile a he end o he documen , a he han being a pa o he igu e ile. Fo each igu e, he ollowing in o ma ion should be p o ided: Figu e numbe (in sequence, using A abic nume als - i.e. Figu e 1, 2, 3 e c); sho i le o igu e (maximum 15 wo ds); de ailed legend, up o 300 wo ds. Please no e ha i is he esponsibili y o he au ho (s) o ob ain pe mission om he copy igh holde o ep oduce igu es o ables ha ha e p e iously been published elsewhe e. P epa ing ables Each able should be numbe ed and ci ed in sequence using A abic nume als (i.e. Table 1, 2, 3 e c.). Tables should also ha e a i le (abo e he able) ha summa izes he whole able; i should be no longe han 15 wo ds. De ailed legends may hen ollow, bu hey should be concise. Tables should always be ci ed in ex in consecu i e nume ical o de . Smalle ables conside ed o be in eg al o he manusc ip can be pas ed in o he end o he documen ex ile, in A4 po ai o landscape o ma . These will be ypese and displayed in he inal published o m o he a icle. Such ables should be o ma ed using he 'Table objec ' in a wo d p ocessing p og am o ensu e ha columns o da a a e kep aligned when he ile is sen elec onically o e iew; his will no always be he case i columns a e gene a ed by simply using abs o sepa a e ex . Columns and ows o da a should be made isibly dis inc by ensu ing ha he bo de s o each cell display as black lines. Commas should no be used o indica e nume ical alues. Colo and shading may no be used; pa s o he able can be highligh ed using symbols o bold ex , he meaning o which should be explained in a able legend. Tables should no be embedded as igu es o sp eadshee iles. La ge da ase s o ables oo wide o a po ai page can be uploaded sepa a ely as addi ional iles. Addi ional iles will no be displayed in he inal, laid-ou PDF o he a icle, bu a link will be p o ided o he iles as supplied by he au ho . Tabula da a p o ided as addi ional iles can be uploaded as an Excel sp eadshee (.xls ) o comma sepa a ed alues (.cs ). As wi h all iles, please use he s anda d ile ex ensions. P epa ing addi ional iles Al hough BMC In ec ious Diseases does no es ic he leng h and quan i y o da a included in an a icle, we encou age au ho s o p o ide da ase s, ables, mo ies, o o he in o ma ion as addi ional iles. Please no e: All Addi ional iles will be published along wi h he a icle. Do no include iles such as pa ien consen o ms, ce i ica es o language edi ing, o e ised e sions o he main manusc ip documen wi h acked changes. Such iles should be sen by email o edi o ial@biomedcen al.com, quo ing he Manusc ip ID numbe . Resul s ha would o he wise be indica ed as "da a no shown" can and should be included as addi ional iles. Since many weblinks and URLs apidly become b oken, BMC In ec ious Diseases equi es ha suppo ing da a a e included as addi ional iles, o deposi ed in a ecognized eposi o y. Please do no link o da a on a pe sonal/depa men al websi e. The maximum ile size o addi ional iles is 20 MB each, and iles will be i us-scanned on submission. Addi ional iles can be in any o ma , and will be downloadable om he inal published a icle as supplied by he au ho . We ecommend CSV a he han PDF o abula da a. Ce ain suppo ed iles o ma s a e ecognized and can be displayed o he use in he b owse . These include mos mo ie o ma s ( o use s wi h he Quick ime plugin), mini-websi es p epa ed acco ding o ou guidelines, chemical s uc u e iles (MOL, PDB), geog aphic da a iles (KML). I addi ional ma e ial is p o ided, please lis he ollowing in o ma ion in a sepa a e sec ion o he manusc ip ex : File name (e.g. Addi ional ile 1) File o ma including he co ec ile ex ension o example .pd , .xls, . x , .pp x (including name and a URL o an app op ia e iewe i o ma is unusual) Ti le o da a Desc ip ion o da a Addi ional iles should be named "Addi ional ile 1" and so on and should be e e enced explici ly by ile name wi hin he body o he a icle, e.g. 'An addi ional mo ie ile shows his in mo e de ail [see Addi ional ile 1]'. Addi ional ile o ma s Ideally, ile o ma s o addi ional iles should no be pla o m-speci ic, and should be iewable using ee o widely a ailable ools. The ollowing a e examples o sui able o ma s. Addi ional documen a ion PDF (Adode Ac oba ) Anima ions SWF (Shockwa e Flash) Mo ies MP4 (MPEG 4) MOV (Quick ime) Tabula da a XLS, XLSX (Excel Sp eadshee ) CSV (Comma sepa a ed alues) As wi h igu e iles, iles should be gi en he s anda d ile ex ensions. Mini-websi es Small sel -con ained websi es can be submi ed as addi ional iles, in such a way ha hey will be b owsable om wi hin he ull ex HTML e sion o he a icle. In 11/3/2014 BMC In ec ious Diseases | Ins uc ions o Au ho s | Resea ch a icles h p://www.biomedcen al.com/bmcin ec dis/au ho s/ins uc ions/ esea cha icle 6/6 o de o do his, please ollow hese ins uc ions: C ea e a olde con aining a s a ing ile called index.h ml (o index.h m) in he oo . Pu all iles necessa y o iewing he mini-websi e wi hin he olde , o sub- olde s. Ensu e ha all links a e ela i e (ie "images/pic u e.jpg" a he han "/images/pic u e.jpg" o "h p://you domain.ne /images/pic u e.jpg" o "C: Documen s and Se ings use name My Documen s mini-websi e images pic u e.jpg") and no link is longe han 255 cha ac e s. Access he index.h ml ile and b owse a ound he mini-websi e, o ensu e ha he mos commonly used b owse s (In e ne Explo e and Fi e ox) a e able o iew all pa s o he mini-websi e wi hou p oblems, i is ideal o check his on a di e en machine. Comp ess he olde in o a ZIP, check he ile size is unde 20 MB, ensu e ha index.h ml is in he oo o he ZIP, and ha he ile has .zip ex ension, hen submi as an addi ional ile wi h you a icle. S yle and language Gene al Cu en ly, BMC In ec ious Diseases can only accep manusc ip s w i en in English. Spelling should be US English o B i ish English, bu no a mix u e. The e is no explici limi on he leng h o a icles submi ed, bu au ho s a e encou aged o be concise. BMC In ec ious Diseases will no edi submi ed manusc ip s o s yle o language; e iewe s may ad ise ejec ion o a manusc ip i i is comp omised by g amma ical e o s. Au ho s a e ad ised o w i e clea ly and simply, and o ha e hei a icle checked by colleagues be o e submission. In-house copyedi ing will be minimal. Non- na i e speake s o English may choose o make use o a copyedi ing se ice. Language edi ing Fo au ho s who wish o ha e he language in hei manusc ip edi ed by a na i e-English speake wi h scien i ic expe ise, BioMed Cen al ecommends Edanz. BioMed Cen al has a anged a 10% discoun o he ee cha ged o BioMed Cen al au ho s by Edanz. Use o an edi ing se ice is nei he a equi emen no a gua an ee o accep ance o publica ion. Please con ac Edanz di ec ly o make a angemen s o edi ing, and o p icing and paymen de ails. Help and ad ice on scien i ic w i ing The abs ac is one o he mos impo an pa s o a manusc ip . Fo guidance, please isi ou page on W i ing i les and abs ac s o scien i ic a icles. Tim Albe has p oduced o BioMed Cen al a lis o ips o w i ing a scien i ic manusc ip . Ame ican Scien is also p o ides a lis o esou ces o science w i ing. Fo mo e de ailed guidance on p epa ing a manusc ip and w i ing in English, please isi he BioMed Cen al au ho academy. Abb e ia ions Abb e ia ions should be used as spa ingly as possible. They should be de ined when i s used and a lis o abb e ia ions can be p o ided ollowing he main manusc ip ex . Typog aphy Please use double line spacing. Type he ex unjus i ied, wi hou hyphena ing wo ds a line b eaks. Use ha d e u ns only o end headings and pa ag aphs, no o ea ange lines. Capi alize only he i s wo d, and p ope nouns, in he i le. All lines and pages should be numbe ed. Au ho s a e asked o ensu e ha line numbe ing is included in he main ex ile o hei manusc ip a he ime o submission o acili a e pee - e iew. Once a manusc ip has been accep ed, line numbe ing should be emo ed om he manusc ip be o e publica ion. Fo au ho s submi ing hei manusc ip in Mic oso Wo d please do no inse page b eaks in you manusc ip o ensu e page numbe ing is consis en be ween you ex ile and he PDF gene a ed om you submission and used in he e iew p ocess. Use he BMC In ec ious Diseases . Foo no es a e no allowed, bu endno es a e pe mi ed. Please do no o ma he ex in mul iple columns. G eek and o he special cha ac e s may be included. I you a e unable o ep oduce a pa icula special cha ac e , please ype ou he name o he symbol in ull. Please ensu e ha all special cha ac e s used a e embedded in he ex , o he wise hey will be los du ing con e sion o PDF. Uni s SI uni s should be used h oughou (li e and mola a e pe mi ed, howe e ). e e ence o ma