Evaluating HBsAg rapid test performance for different biological samples from low and high infection rate settings & populations

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1 Cruz et l. BMC Infectious Diseses (2015) 15:548 DOI /s RESEARCH ARTICLE Evluting HBsAg rpid test performnce for different biologicl smples from low nd high infection rte settings & popultions Open Access Helen Medin Cruz 1, Letici de Pul Sclioni 1, Vness Slete de Pul 2, Elisngel Ferreir d Silv 1, Kyci Mri Rodrigues do Ó 3, Flvio Augusto Pádu Milgres 4, Mrcelo Sntos Cruz 5, Frncisco Inácio Bstos 6, Priscil Pollo-Flores 7, Erotildes Lel 8, An Rit Coimbr Mott-Cstro 9, José Henrique Pilotto 10, Li Lur Lewis-Ximenez 1, Elisbeth Lmpe 1 nd Livi Melo Villr 1,11* Abstrct Bckground: Rpid tests (RTs) might hve severl dvntges over stndrd lbortory procedures, incresing ccess to dignosis, especilly mong vulnerble popultions nd/or those living in remote res. The im of this study ws to evlute the performnce of RTs for the detection of heptitis B virus surfce ntigen (HBsAg) in smples from different popultions/settings. Methods: Three RTs for HBsAg detection (Viki HBsAg, HBsAg Teste Rápido, nd Imuno-Rápido HBsAg ) nd different biologicl specimens (serum, whole blood, nd sliv) were evluted. Anlyses comprised reference pnel nd smples from field studies trgeting suspected cses of heptitis B virus (HBV) (G I), individuls living in deprived res (G II), nd highly vulnerble individuls (G III). Enzyme immunossy (EIA) ws defined s the gold stndrd in this study. Reproducibility, repetbility, nd cross-rectivity with other infectious gents such s dengue, immunodeficiency (HIV), nd heptitis C (HCV) viruses nd T. pllidum were determined. Results: For the reference pnel, the sensitivity nd specificity of ll HBsAg RTs were higher thn %. G I presented the highest kpp vlues for ll rpid ssys using ser smples. When using serum, the sensitivity vlues were higher thn for G I, % for G II nd % for G III, nd the specificity vlues were higher thn for GI, for G II nd % for G III for ll tests. For whole blood smples & the Viki HBsAg ssy, the best performnce ws chieved for GIII (k = %). For sliv smples, the Imuno-Rápido HBsAg ssy showed the highest concordnce vlues with EIA for G I (40.68 %) nd G II (32.20 %). The reproducibility nd repetbility of ll RTs for serum nd sliv were excellent, nd the concordnce between HBsAg EIAs nd RTs using smples rective with other infectious gents vried from % to %. (Continued on next pge) * Correspondence: livifiocruz@gmil.com 1 Lbortory of Virl Heptitis, Oswldo Cruz Institute, FIOCRUZ, Rio de Jneiro, Brzil 11 Present ddress: Virl Heptitis Lbortory, Helio nd Peggy Pereir Pvilion - Ground, Floor - Room B09, FIOCRUZ Av. Brzil, Mnguinhos, Rio de Jneiro, RJ , Brzil Full list of uthor informtion is vilble t the end of the rticle 2015 Cruz et l. Open Access This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( which permits unrestricted use, distribution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 2 of 10 (Continued from previous pge) Conclusions: The overll performnce of RTs for HBsAg in serum ws high/modertely high for ll groups, thereby promoting incresed ccess to HBV dignosis mong vulnerble popultions s well s smples from individuls in emergency settings or remote res. Rpid tests for HBsAg using whole blood could be used in prevlence studies, though these ssys should not be used for sliv smples. Keywords: Heptitis B virus, Rpid tests, Performnce of tests, Dignostic procedures Bckground Exposure to heptitis B virus (HBV) my result in cute nd chronic infections. Two billion individuls re estimted to hve hd contct with the virus nd 240 million to be chronic crriers of HBV. Every yer, pproximtely 600 thousnd people die due to lte complictions of HBV infection [1]. Stndrd HBV dignosis consists of the use of enzyme immunossys (EIAs) nd electrochemiluminescence (ECLIA) with serum or plsm smples [2]. However, these ssys hve limittions tht my compromise their routine use in low- nd middle-income countries: they require trined personnel s well s the vilbility of ll necessry infrstructures. As n lterntive, rpid tests (RTs) my hve severl dvntges over stndrd procedures becuse they re esy to perform nd cn provide conclusive results within few minutes. Additionlly, these tests my be performed on cse-by-cse bsis nd do not require lbortory infrstructure. Moreover, only miniml trining is required to perform RTs [3 5]. Rpid tests for detection of the surfce ntigen of the heptitis B virus (HBsAg) utilize lterl flow device. Different pproches cn be used in lterl flow ssys, but in generl, the ptient s smple is poured over membrne contining two res: the first contins ntibodies ginst HBsAg (nti-hbs) for detection; nd the second, the control re, contins set of regents tht represents the qulity control of the conjugte [4]. HBsAg RTs hve been used for HBV clinicl dignosis nd in serosurveys in different settings nd countries [4 7]. In Brzil, lthough rpid tests for humn immunodeficiency virus (HIV) nd heptitis C virus (HCV) hve been widely used, s recommended by the Brzilin Ministry of Helth (BMoH) [8, 9], no stndrd lgorithm or guideline is yet vilble for HBV RTs. Before the implementtion of rpid testing for HBV dignosis, key prmeters such s their sensitivity, specificity, crossrectivity, reproducibility, nd repetitively should be thoroughly evluted. The determintion of the ccurcy of rpid test compred to gold stndrd dignostic procedure, such s ELISA, is key to minimizing flse positive or negtive results, thus incresing ccess to ccurte dignosis in remote res nd/or emergency settings. The im of this study ws to evlute the performnce of three rpid tests for HBsAg detection using three different types of fluid from individuls in different popultions/settings. Methods Study popultion No comprehensive popultion-bsed serosurvey in Brzil on HBV hs been implemented to dte. Locl/focl studies hve highlighted infection rtes in different segments of the generl popultion, nd different t-risk groups re deeply heterogeneous. A comprehensive pnel of ser from different key groups ws estblished by the uthors. Although bsed on convenience smples, it intentionlly trgeted s mny popultions nd settings s possible, from ll over the country. The reference pnel comprised serum smples obtined from 393 individuls recruited between 2010 nd 2012 t Fiocruz Virl Heptitis Ambultory (Oswldo Cruz Institute, Rio de Jneiro, Brzil), Brzilin Referrl center for the dignosis of virl heptitis (types A, B, C, D nd E). The inclusion criteri for this group were cute, chronic or suspected cses of heptitis B infections, ge of more thn 18 yers nd signed informed consent. Smples from individuls under follow-up t the Fiocruz outptient clinic were tested for HBsAg using two ELISA kits (HBsAg, Rdim, Pomezi, Itly nd ETI-MAK-4, Disorin, Itly) nd three rpid tests (Viki HBsAg, Biomérieux, Frnce; HBsAg Teste Rápido, Doles, Brzil, nd Imuno- Rápido HBsAg, Wm, Brzil). A field study ws composed of three groups (I-III), with ech prticipnt providing serum, whole blood nd/ or sliv. The serum smples were tested for HBsAg using one ELISA kit (ETI-MAK-4, Disorin, Itly), nd ll biologicl smples were ssyed for ll RTs evluted. Group I (G I) comprised 371 individuls referred to Fiocruz Virl Heptitis Ambultory (Oswldo Cruz Institute, Rio de Jneiro) from 2009 to Inclusion criteri were ttendnce t Fiocruz Virl Heptitis Ambultory, residing in underserved nd impoverished res in Rio de Jneiro City (cpitl of Rio de Jneiro Stte) nd suspected cse of virl heptitis infection. This group ws considered the high-risk group. Group II (G II) comprised 881 individuls living in three (of the five) Brzilin mcro-regions (Southest, North nd Midwest) nd belonging to the generl

3 Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 3 of 10 popultion, mong which HBV prevlence hs been low in recent yers. None of these individuls were recruited in virl heptitis mbultory cre settings, nd this group ws considered to be low risk for HBV. These smples were obtined from individuls living in Rio de Jneiro Stte (Petrópolis nd Mcé cities), Tocntis Stte (Tocntinópolis city), nd Mto Grosso do Sul Stte (the Pntnl region) in nd who greed to be tested within the context of public cmpigns iming to increse virl heptitis dignosis nd the prompt referrl of infected ptients for tretment nd cre. The individuls from Mto Grosso do Sul Stte lived in communities of the Pntnl region, up to 385 km fr from Cmpo Grnde City (Mto Grosso do Sul Stte), wheres other individuls lived up to 217 km fr (by river trnsporttion) from the city of Corumbá (Mto Grosso do Sul Stte). The individuls from Tocntins Stte lived in rurl communities from Tocntinópolis, 30 km fr wy from the urbn re of the city. Such individuls belong to socilly isolted popultions living in deprived, underserved communities. The individuls from Rio de Jneiro Stte were employees from privte hospitl locted in Petrópolis city who belonged to the middle-clss s well s individuls living in underprivileged communities of Mcé city. Petrópolis city is locted in mountin region, nd Mcé city is situted in the northern region of Rio de Jneiro Stte. Our im in this study ws to ssess s mny individuls s possible from remote res nd/or deprived communities, s well s smll subgroup of people from the middle-clss strtum. This subgroup flls short of popultion-bsed repository, but deliberte efforts were mde to estblish pool s diverse s possible, focusing on underserved popultions tht could benefit the most from extended testing strtegies. Finlly, Group III (G III) ws composed of 251 vulnerble individuls, including 158 beuticins nd 93 hevy users of crck cocine from Rio de Jneiro Stte. All study prticipnts nd/or their legl gurdins consented nd signed informed consent forms prior to enrollment. Ethicl pprovl for the study ws issued by the Oswldo Cruz Foundtion Ethics Committee. Lbortory results were promptly returned to the ptients physicins. Smple collection Whole blood nd serum smples were collected by venipuncture using vcutiner tubes, with (BD Vcutiner contining the nticogulnt EDTA) nd without (BD SST II Advnce ) dditives, respectively. Sliv smples were collected using commercil collection devices (Slivette ; Srstedt, Germny) nd were mixed with 1 ml of trnsport buffer. Orl fluid smples were centrifuged (1,400 g for 10 min) nd stored t 20 C until ssyed, s detiled elsewhere [10]. For the reference pnel, only serum smples were ssyed, wheres serum, whole blood nd sliv the field studies were nlyzed. HBsAg nd HBV detection Serum smples from the reference pnel were tested for HBsAg mrkers using two commercil EIAs (HBsAg, Rdim, Pomezi, Itly nd ETI-MAK-4, Disorin, Itly) following the mnufcturers instructions. Only smples with concordnt results defined by both ssys were included in the study. Serum smples from the field study were ssyed for HBsAg detection using commercil EIA (HBsAg, Rdim, Pomezi, Itly). All HBsAg-rective smples in the EIA were retested in duplicte. All serum smples were ssyed for totl ntibodies directed ginst the totl core ntigen (nti-hbc totl) s well s nti-hbs using EIAs (Disorin, Itly). Serum smples were lso ssyed for nti-hbc IgM, HBV e ntigen (HBeAg) nd ntibodies ginst HBeAg (nti-hbe) using commercil EIAs nd ECLIAs (Disorin, Itly) when sufficient smple volume ws vilble. Rpid test evlution Three HBsAg rpid tests were evluted: Viki HBsAg (Biomerieux, Frnce), HBsAg Teste Rápido (Doles, Brzil), nd Imuno-Rápido HBsAg (Wm, Brzil). All the RTs re pproved by the Brzilin Ntionl Helth Surveillnce Agency (ANVISA), which is responsible for the regultion, control nd supervision of products nd services tht involve risk to public helth. Viki HBsAg hs CE IVD pprovl but does not hve FDA pprovl or WHO registrtion. Imuno-Rápido HBsAg does not hve FDA pprovl or WHO registrtion; such informtion is not vilble for HBsAg Teste Rápido. All three tests re qulittive tests bsed on immunochromtogrphic techniques for lterl ssocition of monoclonl nd polyclonl ntibodies specific for HBsAg. The Viki HBsAg llows the detection of the min d nd y subtypes in serum, plsm nd whole blood by dding 75 μl of ech smple to the test pltform. Cpillry blood by fingerstick cn lso be used in this test. According to the mnufcturers instructions, the nlyticl sensitivity of the RTs is less thn or equl to 2 IU/ ml for Viki HBsAg nd from 10 IU/ml for HBsAg Teste Rápido nd Imuno-Rápido HBsAg. Redings were vilble within 15 min (though for negtive smples, it ws necessry to wit up to 30 min to confirm the result). The Imuno-rápido HBsAg nd HBsAg teste rápido tests llow the detection of HBsAg

4 Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 4 of 10 in serum smples, nd both ssys use 100 microliters of smple. The results cn be ssessed within 20 min. All procedures for rpid tests were performed ccording to the mnufcturers recommendtions, except for the sliv smples, for which twofold increses in smple volume (i.e., 75 μl 150 μl nd 100 μl 200 μl) were dopted to increse the sensitivity of HBsAg detection. The mnufcturers recommendtions do not include the nlysis of sliv smples. However, previous ttempts mde by our reserch group with regrd to heptitis C [11] were successful nd motivted the current ttempt. In the present study, we extended sliv nlyses by incorporting HBsAg detection. Serum nd sliv were ssyed for ll HBsAg RTs, nd whole blood smples were evluted using the Viki HBsAg test becuse it is the only test specificlly designed for the ltter. Reproducibility nd repetbility To evlute the reproducibility nd repetbility of HBsAg rpid tests, four smples (2 serum nd 2 sliv smples) were tested in eleven replictes, ech by two different opertors, for two consecutive dys. One HBsAgrective nd nother HBsAg-non-rective serum smple by EIA were included. HBV-negtive individuls donted sliv smples. These sliv smples were then diluted (1:1) with n HBV-rective serum smple. The HBsAg rpid testing procedures were similr to the procedures described bove for serum nd sliv. kpp sttistics were used to cross-compre the results of rpid tests nd EIA. Cross-rectivity studies Serum smples rective for other infectious gents were included in the nlysis to ssess the cross-rectivity of the HBsAg rpid tests. Twenty serum smples rective for dengue virus (five for ech of the co-circulting serotypes: DENV-1, DENV-2, DENV-3 nd DENV-4), 69 HIV-rective serum smples, 49 Treponem pllidumrective serum smples, nd 137 HCV-rective smples were included. HBsAg ws ssyed using commercil EIA (HBsAg One, RADIM) nd HBsAg rpid tests (Viki HBsAg; HBsAg Teste Rápido, nd Imuno- Rápido HBsAg ). Dt nlysis The dt nlysis comprised smples with well-defined serology. Indeterminte smples detected by EIA were excluded. Socio-demogrphic, epidemiologicl, clinicl, EIA nd rpid test results were entered into n Access dtbse. Sttisticl nlyses were performed using SPSS 20.0 for Windows (SPSS Inc., USA). Prmeters ssocited with test performnce were evluted using GrphPd InStt Progrms, version 3.01 (GrphPd Softwre, Sn Diego, USA) nd MedClc, version (MedClc Softwre,Mri-kerke,Belgium). The RT results were cross-compred with the EIA results, which ws defined for the ske of this study s the gold stndrd. Anlytic ctegories were defined s follows: true positive results (TP positive in both tests), true negtive results (TN negtive in both tests), flse positive results (FP positive in RT nd negtive in commercil EIA), flse negtive results (FN negtive in RT nd positive in commercil EIA). The clinicl sensitivity (Cs), specificity (S), positive predictive vlue (PPV), nd negtive predictive vlue (NPV) for ech rpid test were evluted, nd their respective 95 % confidence intervls (95 % CI) were clculted. Contingency tbles nd respective sttistics were used to cross-compre findings from different testing procedures nd popultions/settings. Concordnce between the pnel s results nd the results from rpid tests ws ssessed by kpp sttistics [12]. P-vlues (two-tiled) <0.05 were considered sttisticlly significnt. Results HBsAg rpid test performnce using reference pnels The reference pnel ws composed of 393 individuls, 103 of which were HBsAg rective (ser), wheres 290 smples did not show HBsAg ccording to EIAs. The men ge (± stndrd devition) of the ptients ws yers (±14.78), nd most were femle (61.24 %). HBsAg ws detected in 101, 98 nd 96 of the smples, with sensitivities of 98.06, nd % by Viki HBsAg, Imuno-Rápido HBsAg nd HBsAg Teste Rápido, respectively (Tble 1). According to the three RT ssys, HBsAg flse negtive serum smples hd low opticl density/cut-off vlue rtio (OD/CO) by EIA when compred to HBsAg true positive smples (concise informtion bout the nlyticl detection limits is vilble in Additionl file 1: Web Appendix 1). Most of the HBsAg-rective smples lso presented nti-hbc totl, wheres nti-hbc nd nti-hbs were not detected in most of the HBsAg-non-rective smples, independent of the group studied (21.60 to %). In ddition, nti-hbc IgM, HBeAg, nti-hbe were most frequently detected in the reference pnel nd group I (the serologicl chrcteristics of HBV mrkers is vilble in Additionl file 1: Web Appendix 2). HBsAg rpid test performnce using field smples Overll, 3,273 biologicl smples were collected in the different field studies, nd 1,503 serum, 1,268 whole blood nd 502 sliv smples were included in the nlyses. The men ge of the individuls ws (±18:43) yers, (±12.95) yers nd (±16:15)

5 Tble 1 Accurcy metrics (point estimtes nd 95%CIs) of three rpid tests compred to results of HBsAg One nd ETI-MAK-4, enzyme immunossys Mnufcturer TP FN TN FP Sensitivity Specificity PPV NPV K (CI%) HBsAg non-rective/hbsag rective (n = 393) Viki HBsAg % ( ) % ( ) % ( ) % ( ) % ( ) Imuno-Rápido HBsAg % ( ) % ( ) ( ) % ( ) 94.7 % ( ) HBsAg teste rápido % ( ) % ( ) % ( ) % ( ) % ( ) Legends: TP True positive, FN Flse negtive, TN True negtive, FP Flse positive, PPV Positive Predictive Vlue, NPV Negtive Predictive Vlue, k Kpp sttistics, n number of observtions (biologicl smples), CI confidence intervl Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 5 of 10

6 Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 6 of 10 yers for those donting serum, whole blood nd sliv, respectively. Most individuls were women (51.93 % for serum, % for whole blood, nd % for sliv). The number of serum, whole blood nd sliv smples from the different groups cn be summrized s follows: G I - 371, 108 nd 185 individuls; G II - 881, 767 nd 160 individuls; G III - 251, 393 nd 157 individuls, respectively. Overll (ll groups nd serum smples), Viki HBsAg presented the highest kpp vlue (96.08 %), followed by HBsAg teste rápido (88.41 %) nd Imunorápido HBsAg (87.62 %). The specificities of ll RTs were higher thn 97 %, nd Viki HBsAg presented the highest sensitivity (Tble 2). For serum smples from G I, the sensitivity ws 93.41, 94.01, nd % nd specificity 99.51, 99.51, nd % using HBsAg teste rápido, Imuno-rápido HBsAg nd Viki HBsAg, respectively. For smples from G II, sensitivity ws % for ll tests, nd specificity ws 97.83, 97.26, nd % for HBsAg teste rápido, Imuno-rápido HBsAg nd Viki HBsAg, respectively. With respect to G III, sensitivity ws % for ll tests, nd specificity ws 99.18, 99.59, nd % for HBsAg teste rápido, Imuno-rápido HBsAg nd Viki HBsAg, respectively. As observed in the reference pnel, HBsAg flse negtive smples, s defined by RTs, presented low vlues of OD/CO by EIA compred to true HBsAg-positive smples (Additionl file 1 vilble in Web Appendix 1). The Viki HBsAg test performed using whole blood smples presented the highest kpp vlue for G III (79.75 %), followed by G I (72.7 %). Concordnce ws not determined mong the G II smples becuse the test could not detect true positives. Immuno-Rápido HBsAg demonstrted the highest kpp vlue with EIA for G I (40.68 %) sliv smples, followed by G III (32.20 %). For G III smples, none of the vilble RTs could detect true HBsAg-positive smples; thus, concordnce vlue could not be clculted (Tble 3). Reproducibility nd repetbility Reproducibility nd repetbility nlyses comprised 528 tests (264 serum nd 264 sliv smples). The HBsAg teste rápido, Immuno-Rápido HBsAg nd Viki HBsAg ssys exhibited % kpp vlue for the EIA results for ll smples (i.e., serum nd sliv smples HBsAg rective/non-rective). Cross-rectivity with other infectious gents HIV-, dengue virus-, HCV- nd T. pllidum-rective smples were evluted using HBsAg rpid tests nd EIA. HBsAg-rective results were found mong eight HCV smples, nine T. pllidum smples, nd 15 HIV smples using EIA. In contrst, no HBsAg-rective smple ws found mong the DENV smples. The kpp sttistic for EIA nd RTs for HBsAg detection vried from % to % for T. pllidum, % to % for HIV, % to % for HCV nd 100 % for dengue for ll mnufcturers. Flse negtive results were found for ll mnufcturers for T. pllidum, HIV nd HCV, nd flse positive results were found in one HIV-rective smple (using HBsAg teste rápido ) nd two HCV-rective smples (one by HBsAg teste rápido nd the other by Immuno-Rápido HBsAg ) (Tble 4). Discussion The present study demonstrtes the usefulness of HBsAg rpid tests in both the lb nd the field. All rpid tests detected HBsAg mong serum smples from reference pnel, with high sensitivity nd specificity within contexts of high bckground prevlence. However, sensitivity ws found to be poor when the bckground prevlence ws low. In the field study, HBsAg rpid tests demonstrted higher thn % specificity mong serum smples, regrdless of the mnufcturer nd the group under study, demonstrting the bility of these ssys to detect HBsAg true negtive smples [5, 12 15). The performnce of RTs could be influenced by the ntigen concentrtion becuse flse negtive smples hd low verge vlues of OD/CO compred to true positive smples, in greement with previous studies [4, 14]. In the present study, the presence of HBV mrkers, such s nti-hbc or nti-hbs, did not pper to influence the HBsAg RT results. However, it ws not possible to evlute ll the smples for HBeAg, nti-hbe nd/or nti-hbc IgM due to low smple volumes. Thus, it ws not possible to evlute the influence of these mrkers on the performnce of HBsAg RT performnce. The Viki HBsAg test presented the best performnce in both the lbortory nd field for serum smples, nd the highest concordnce ws observed mong G I ptients. Such optiml performnce my be secondry to the high HBsAg prevlence in this group, s observed by Lien et l. [13] using RTs from other mnufcturers. In Brzil, confirmed HBV cses from 1999 to 2011 ( ) were most reported in the southest region (36.30 %), where Rio de Jneiro is locted (16). Low HBsAg prevlence could influence the performnce of rpid tests, s found for the G II nd G III smples. In Brzil, the HBsAg prevlence vries from 0.63 % in the northern region to 0.31 % in both the southestern nd midwestern regions [16]. Previous studies conducted mong beuticins nd crck users showed lower HBsAg prevlence of 0 % to 6.2 % in Rio de Jneiro [17, 18]. However, in the present study the prevlence in these groups ws 2.3 %, corroborting the priori ssumption these groups re prticulrly vulnerble to HBV infection.

7 Tble 2 Accurcy metrics (point estimtes nd 95%CIs) of three rpid tests compred to results obtined by enzyme immunossy ETI-MAK-4 in serum smples ccording to the chrcteristics of the study popultion Profile/Mnufcturer (n) TP FN TN FP Sensitivity Specificity PPV NPV K Group I Viki HBsAg (371) % ( ) % ( ) % ( ) % ( ) % ( ,98) Imuno-rápido HBsAg (371) % ( ) % ( ) % ( ) % ( ) % ( ) HBsAg Teste rápido (371) % ( ) % ( ) % ( ) % ( ) % ( ) Group II Viki HBsAg (881) % ( ) % ( ) % ( ) % ( ) % ( ) Imuno-rápido HBsAg (881) % ( ) % ( ) % ( ) % ( ,97) % ( ) HBsAg Teste rápido (881) % ( ) % ( ) % ( ) % (99, ) % ( ) Group III Viki HBsAg (251) % ( ) % ( ) % ( ) % ( ) % ( ) Imuno-rápido HBsAg (251) % ( ) % ( ) % ( ) % ( ) % ( ) HBsAg Teste rápido (251) % ( ) % ( ) % ( ) % ( ) % ( ) Overll Viki HBsAg (1503) % ( ) % ( ) % ( ) % ( ) % ( ) Imuno-rápido HBsAg (1503) % ( ) % ( ) % ( ) % ( ) % ( ) HBsAg Teste rápido (1503) % ( ) % ( ) % ( ) % ( ) % ( ) Legends: TP True positive, FN Flse negtive, TN True negtive, FP Flse positive, PPV Positive Predictive Vlue, NPV Negtive Predictive Vlue, k kpp sttistics, n number of smples, CI confidence intervl Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 7 of 10

8 Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 8 of 10 Tble 3 Kpp sttistics nd positive nd negtive smples detected by rpid tests using sliv (3) nd whole blood (1) smples compred to results obtined using respective serum smples by enzyme immunossy ETI-MAK-4, ccording to the chrcteristics of the popultion under nlysis Profile/Mnufctory Biologicl specimen TP FN TN FP K Group I Viki HBsAg - SALIVA % ( ) Imuno-Rápido HBsA - SALIVA % ( ) Teste rápido HBsAg - SALIVA % ( ) Viki HBsAg - WHOLE BLOOD % ( ) Group II Viki HBsAg - SALIVA Imuno-Rápido HBsA - SALIVA % ( ) Teste rápido HBsAg - SALIVA Viki HBsAg - WHOLE BLOOD Group III Viki HBsAg - SALIVA Imuno-Rápido HBsA - SALIVA Teste rápido HBsAg - SALIVA Viki HBsAg - WHOLE BLOOD % ( ) Overll Viki HBsAg - SALIVA % (0,00 25,11) Imuno-Rápido HBsA - SALIVA % ( ) Teste rápido HBsAg - SALIVA % ( ) Viki HBsAg - WHOLE BLOOD % ( ) Legend: TP True positive, FN Flse negtive, TN True negtive, FP Flse positive, k kpp sttistics, n number of observtions (biologicl smples), CI confidence intervl not menble to clcultion The best performnce of the Viki HBsAg test in whole blood smples ws observed in the G III smples, fvoring the pplicbility of this rpid test to vulnerble individuls. Nonetheless, the kpp vlue ws reltively low (72.73 %) compred to previous study conducted in Frnce (96.98 %) [4]. The observed difference my be secondry to different blood collection procedures: blood ws obtined by venipuncture in tubes without nticogulnt in the previous study [4] yet by venipuncture in tubes with nticogulnt in the present study. The best performnce for sliv smples ws chieved using the Immuno-Rápido HBsAg ssy in the G I nd II smples. However, high number of flse negtive results were observed for the tests from ll mnufcturers, regrdless of the group under study, which is most likely secondry to the low concentrtion of HBsAg in sliv smples [19]. In ddition, HBsAg rpid tests were not originlly developed for sliv smples, which my lso explin the low concordnce with EIA results. Accordingly, our findings must be viewed s wrning ginst the use of these procedures for HBsAg detection in sliv smples. The previous promising findings respecting heptitis C testing were, unfortuntely, not observed for HBsAg. In the present study, the volume of sliv smple ws incresed in ll rpid tests to improve the sensitivity of the ssy, but this procedure could not be trnslted into concrete benefits. Additionl modifictions, such s longer incubtion period, my be pursued by future studies. The HBsAg rpid tests demonstrted excellent repetbility nd reproducibility in serum nd rtificilly contminted sliv smples, demonstrting the good performnce of these ssys under lbortory conditions. Although two met-nlysis studies showed high pooled ccurcy for RTs for HBsAg [20, 21], Khuroo et l. [21] lso observed wide vrition in sensitivity mong individul tests (43.5 % to 99.8 %), which could be due to the design of the studies or popultion chrcteristics becuse the performnce of RTs re better in developed thn in developing countries. In the present study, wide vrition of sensitivity of RTs for HBsAg ws observed ccording to the group studied (60.00 % to %), demonstrting the importnce of evluting RTs mong specific popultions before implementtion t lrge scle. Regrding cross-rectivity, the best results using serum smples were found mong rective dengue smples, most likely due to the bsence of HBsAg-rective smples

9 Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 9 of 10 Tble 4 Kpp sttistics nd positive nd negtive smples detected by HBsAg rpid tests mong serum smples with rective serology for different infections compred to results obtined by enzyme immunossy ETI-MAK-4 TP (n) FN (n) FP (n) TN (n) K% (IC%) Dengue (n = 20) Viki HBsAg % Imuno-Rápido HBsAg % Teste rápido HBsAg % T. pllidum (n = 49) Viki HBsAg % Imuno-Rápido HBsAg % Teste rápido HBsAg % HIV (n = 69) Viki HBsAg % Imuno-Rápido HBsAg % Teste rápido HBsAg % HCV (n = 137) Viki HBsAg % Imuno-Rápido HBsAg % Teste rápido HBsAg % Legend: TP True positive, FN Flse negtive, TN True negtive, FP Flse positive, k kpp sttistics, n number of observtions (biologicl smples), CI confidence intervl nd/or the low number of dengue smples under nlysis (i.e., due to bet error). Regrdless, for T. pllidum-, HIVnd HCV-rective smples, flse negtive nd positive HBsAg results were observed for ll RTs. However, the poorest performnce ws observed mong HIV-rective smples. This is of concern becuse under rel life conditions, non-negligible frction of ptients my be co-infected by the two viruses, which my yield confusing results nd inconclusive clinicl interprettions. The Viki HBsAg nd HBsAg teste rápido tests showed better kpp vlues for HIV- nd HCV-rective smples, respectively, nd both Viki HBsAg nd HBsAg teste rápido showed better kpp vlues for T. pllidum-rective smples. These results suggest tht both ssys my hve good performnce with smples rective for other infections. Other HBsAg RTs presented good performnce mong HIV-rective smples [6, 7]. In ddition, met-nlysis demonstrted tht co-infections (for exmple HIV, HCV, tuberculosis) did not influence the dignostic ccurcy of HBsAg RTs, which could be helpful for the doption of these ssys in endemic res where these co-infections re highly prevlent [21]. This study presents some limittions, such s the bsence of HBsAg neutrliztion, HBV DNA testing or HBsAg concentrtion due to low smple volume. However, rective HBsAg smples were retested in duplicte to confirm the HBsAg results. Conclusion In conclusion, the present study showed moderte to high concordnce of HBsAg rpid tests using serum smples from different popultions/settings. These findings could be useful for HBV dignosis mong individuls who re highly vulnerble to HBV infection s well s those recruited from emergency settings or remote res. In ddition, sliv smples should not be used for HBsAg detection with the ssys evluted in the present study. Additionl file Additionl file 1: Appendix 1. Men vlues of OD/CO from EIE mong flse negtives nd true positive rpid tests for HBsAg detection in three rpid tests ccording to the chrcteristics of the study popultion. Appendix 2. HBV mrkers (nti-hbc, nti-hbs, nti-hbc IgM, HBeAg, nti-hbe) detected in serum smples using enzyme immunossy ccording to the popultion studied. (DOC 63 kb) Abbrevitions Anti-HBc IgM: Antibodies directed ginst the core ntigen IgM; Anti-HBc totl: Antibodies directed ginst the core ntigen; Anti-HBe: Antibodies ginst HBeAg; ANVISA: Ntionl Helth Surveillnce Agency; Anti-HBs: Antibodies directed ginst heptitis B surfce ntigen; BMoH: Brzilin Ministry of Helth; CI: Confidence intervl; CO: Cut-off vlue; Cs: Clinicl sensitivity; ECLIA: Electrochemiluminescence; EIA: Enzyme immunossy; FDA: Food nd drug dministrtion; FN: Flse negtive result (negtive in RT nd positive in commercil EIA); FP: Flse positive result (positive in RT nd negtive in commercil EIA); HBeAg: HBV e ntigen; HBsAg: Surfce ntigen of the heptitis B virus; HBV: Heptitis B virus; HCV: Heptitis C virus; HIV: Humn immunodeficiency virus; NPV: Negtive predictive vlue; OD: Opticl density; PPV: Positive predictive vlue; S: Specificity; TN: True negtive result (negtive in both RT nd EIA); TP: True positive result (positive in both RT nd EIA). Competing interests The uthors disclose no ctul or potentil conflict of interest, including ny finncil, personl or other reltionships with people or orgniztions, within two yers of the beginning of this study tht could inppropritely influence the study. Authors contributions LMV nd EL conceived the study; LMV, HMC nd EL designed the study protocol; LLLX, MSC, FIB, KMRO, FAPM, PPF, ErL, ARCMC, JHP crried out the clinicl ssessment, subject selection nd recruitment; HMC, LPS, VSP, EFS, LMV performed the rpid tests, immunossys, nd nlysis nd interprettion of these dt; LMV, HMC, FIB drfted the mnuscript; FIB, VSP, LMV, LLLX, EL criticlly revised the mnuscript for intellectul content. All uthors red nd pproved the finl mnuscript. Acknowledgments The uthors wish to thnk Julin Custódio Miguel, Rent Tourinho dos Sntos, Jqueline Correi de Oliveir for technicl ssistnce in the smple collection. This reserch ws supported by the Support Foundtion for Reserch in Rio de Jneiro Stte (FAPERJ), Brzilin Ntionl Council of Technologicl nd Scientific Development (CNPq) nd the Oswldo Cruz Foundtion (FIOCRUZ). Author detils 1 Lbortory of Virl Heptitis, Oswldo Cruz Institute, FIOCRUZ, Rio de Jneiro, Brzil. 2 Lbortory of Technologicl Development of Virology, Oswldo Cruz Institute, FIOCRUZ, Rio de Jneiro, Brzil. 3 São Lucs Hospitl, Petropolis, Rio de Jneiro, Brzil. 4 Medicine Fculty, Federl University of Tocntins, Plms, Brzil. 5 Institute of Psychitry, Federl University of Rio de

10 Cruz et l. BMC Infectious Diseses (2015) 15:548 Pge 10 of 10 Jneiro, Rio de Jneiro, Brzil. 6 Institute of Communiction nd Scientific Informtion & Technology for Helth, Oswldo Cruz Foundtion, Rio de Jneiro, Brzil. 7 Antonio Pedro University Hospitl, Federl Fluminense University, Rio de Jneiro, Brzil. 8 Federl University of Rio de Jneiro, Cmpus Mcé, Rio de Jneiro, Brzil. 9 Federl University of Mto Grosso do Sul nd FIOCRUZ-MS, Cmpo Grnde, MS, Brzil. 10 Lbortory of AIDS nd Moleculr Immunology, Oswldo Cruz Institute, FIOCRUZ, Rio de Jneiro, Brzil. 11 Present ddress: Virl Heptitis Lbortory, Helio nd Peggy Pereir Pvilion - Ground, Floor - Room B09, FIOCRUZ Av. Brzil, Mnguinhos, Rio de Jneiro, RJ , Brzil. Received: 23 My 2015 Accepted: 28 October Villr LM, de Pul VS, de Almeid AJ, KM d Ó, Miguel JC, Lmpe E. Knowledge nd prevlence of virl heptitis mong beuticins. J Med Virol. 2014;86: Prry JV, Perry KR, Mortimer PP. Sensitive tests for virl ntibodies in sliv; n lterntive to tests on serum. Lncet. 1987;2: Shivkumr S, Peeling R, Jfri Y, Joseph L nd Pi NP. Rpid Point-of-Cre First-Line Screening Tests for Heptitis B Infection: A Met-Anlysis of Dignostic Accurcy ( ). Am J Gstroenterol dvnce online publiction, 2012; doi: /jg Khuroo MS, Khuroo NS, Khuroo MS. Accurcy of rpid point-of-cre dignostic tests for heptitis b surfce ntigen systemtic review nd met-nlysis. J Clin Exp Heptol. 2014;4(3): References 1. World Helth Orgniztion. Heptitis B medicentre/fctsheets/fs204/en/ [Accessed on ]. 2. Gerlich WH. Medicl Virology of Heptitis B: how it begn nd where we re now. Virol J. 2013;10: Seremb E, Ocm P, Opio CK, Kgimu M, Yun HJ, Attr N, et l. Vlidity of the rpid strip ssy test for detecting Hbsg in ptients dmitted to hospitl in Ugnd. J Med Virol. 2010;82: Bottero J, Boyd A, Gozln J, Lemoine M, Crrt F, Collignon A, et l. Performnce of rpid tests for detection of HBsAg nd Anti-HBsAb in lrge cohort. Frnce J Heptol. 2013;58: Chevliez S, Chlline D, Nij H, Luu TC, Lperche S, Ndl L, et l. Performnce of new rpid test for the detection of heptitis B surfce ntigen in vrious ptient popultions. J Clin Virol. 2014;59: Dvies J, vn Oosterhout JJ, Nyirend M, Bowden J, Moore E, Hrt IJ, et l. Relibility of rpid testing for heptitis B in region of high HIV endemicity. Trns R Soc Trop Med Hyg. 2010;104: Frnzeck FC, Ngwle R, Msongole B, Hmisi M, Abdul O, Henning L, et l. Virl heptitis nd rpid dignostic test bsed screening for HBsAg in HIV-infected ptients in rurl Tnzni. PLoS One. 2013;8(3), e Brzilin Helth Ministry. Technicl mnul for dignosis of HIV infection /_p_mnul_tecnico_hiv_finl_pdf_p pdf. [Accessed on ]. 9. Brzilin Helth Ministery. Include nd chnge procedures of procedures tble, Drugs, Orthotics, Prosthetics nd Specilty Mterils for SUS. Ordinnce n. 730 from July 02 nd sudelegis/ss/2013/prt0730_02_07_2013.html. [Accessed on ]. 10. Cruz HM, d Silv EF, Villel-Nogueir CA, Nbuco LC, KM d Ó, Lewis-Ximenez LL, et l. Evlution of sliv specimens s n lterntive smpling method to detect heptitis B surfce ntigen. J Clin Lb Anl. 2011;25: Sclioni Lde P, Cruz HM, de Pul VS, Miguel JC, Mrques VA, Villel-Nogueir CA, et l. Performnce of rpid heptitis C virus ntibody ssys mong high- nd low-risk popultions. J Clin Virol. 2014;60(3): Altmn DG. Prcticl Sttistics for Medicl Reserch. London: Chpmn nd Hll; Lien TX, Tien NT, Chnpong GF, Cuc CT, Yen VT, Soderquist R, et l. Evlution of rpid dignostic tests for the detection of humn immunodeficiency virus types 1 nd 2, heptitis B surfce ntigen, nd syphilis in Ho Chi Minh City, Vietnm. Am J Trop Med Hyg. 2000;62: Mrtínez-Lms L, Rodríguez JJ, Regueiro BJ, Aguiler A. Evlución de um test rápidominmunocromtográfico pr l detección del ntígeno de superficie de l heptitis B. Rev Esp Quimioter. 2011;24: Rndrimin F, Crod JF, Rtsim E, Chrétien JB, Richrd V, Tlrmin A. Evlution of the performce of four rpid tests for detection of heptitis B surfce ntigen in Antnnrivo, Mdgscr. J Virol Methods. 2008;151: Brzilin Helth Ministry. Secretrit of Helth Surveillnce. STD, Aids nd Virl Heptitis Deprtment. Epidemiologicl Bulletin Virl Heptitis /boletim_epidemiol_gico_heptites_viris_2012_ve_12026.pdf. [Accessed on ]. 17. Sntos Cruz M, Andrde T, Bstos FI, Lel E, Bertoni N, Villr LM, et l. Key drug use, helth nd socio-economic chrcteristics of young crck users in two Brzilin cities. Int J Drug Policy. 2013;24: Submit your next mnuscript to BioMed Centrl nd tke full dvntge of: Convenient online submission Thorough peer review No spce constrints or color figure chrges Immedite publiction on cceptnce Inclusion in PubMed, CAS, Scopus nd Google Scholr Reserch which is freely vilble for redistribution Submit your mnuscript t