Aott RelTime 2N40 51-608234/R1 HBV Customer Servie: 1-800-553-7042 This pkge insert must e red refully prior to use. Pkge insert instrutions must e followed ordingly. Reliility of ssy results nnot e gurnteed if there re ny devitions from the instrutions in this pkge insert. List Numer Lot Numer In Vitro Dignosti Medil Devie Store t -10 C or older Mnufturer Consult instrutions for use Expirtion Dte CAUTION: Hndle humn soured mterils s potentilly infetious. Consult instrutions for use. (Infetion Risk) Key to symols used Clirtor A Clirtor B Negtive Control Low Positive Control High Positive Control Internl Control Amplifition Regent Pk See REAGENTS setion for full explntion of symols used in regent omponent nming. NAME Aott RelTime HBV Assy INTENDED USE Aott RelTime HBV ssy is n in vitro polymerse hin retion (PCR) ssy for use with the Aott m2000 System DNA regents nd with the Aott m2000sp nd m2000rt instruments for the quntittion of Heptitis B Virus (HBV) DNA in humn serum or plsm (EDTA) from hronilly HBV-infeted individuls. The ssy is intended for use s n id in the mngement of ptients with hroni HBV infetion undergoing nti-virl therpy. The ssy n e used to mesure HBV DNA levels t seline nd during tretment to id in ssessing response to tretment. The results from the Aott RelTime HBV ssy must e interpreted within the ontext of ll relevnt linil nd lortory findings. Assy performne for determining the linil stge of HBV infetion hs not een estlished. Clinil performne hrteristis hve een estlished for individuls treted with defovir dipivoxil. This ssy is not intended for use s sreening test in lood or lood produts for HBV or s dignosti test to onfirm the presene of HBV infetion. SUMMARY AND EXPLANATION OF THE TEST HBV is smll, irulr, prtilly doule-strnded DNA virus of pproximtely 3,200 se pirs. 1 The virus n use lifelong infetion, irrhosis (srring) of the liver, liver ner, liver filure, nd deth. 2 The prevlene of HBV infetion nd the method of trnsmission vry gretly round the world. In ountries with high prevlene of hroni HBV infetion, the most ommon route of infetion is from mother-to-hild t irth or from hild-to-hild during erly hildhood. In res of low prevlene, infetion is usully quired during dulthood through intrvenous drug use or high-risk sexul tivity. 3 The risk of developing hroni HBV infetion from ute exposure rnges from 90% in neworns of HBV-infeted mothers to 25-30% for hildren under 5 nd less thn 10% in dults. 1 Immuniztion is the most effetive wy to prevent HBV infetion, nd n offer greter thn 95% protetion ginst the development of hroni infetion. 3 Quntittion of HBV DNA is importnt in the evlution nd mngement of ptients with hroni HBV infetion. Current guidelines reommend HBV virl lod to determine whih hroni HBV ptients should e treted nd to monitor their response to therpy. 4,5,6 A low seline virl lod hs een shown to e preditive of response to therpy. 5 Conversely, high seline virl lod is preditive of resistne to therpy s well s relpse following therpy, nd hs lso een found to e n independent risk ftor for heptoellulr rinom. 7 Current tretment options inlude interferon, peginterferon, nd ntivirl drugs suh s lmivudine, defovir, nd tenofovir. 5,6,8 HBV DNA in serum or plsm n e quntitted using nulei id mplifition or signl mplifition tehnologies. 9 The Aott RelTime HBV ssy uses PCR tehnology omined with homogeneous rel time fluoresent detetion for the 1 quntittion of HBV DNA. The seletion of highly onserved region in the Surfe gene provides for the detetion of HBV genotypes A, B, C, D, E, F, G, nd H. The lotion of the trget region in the N terminl third of the Surfe gene ensures tht the ssy is not impted y YMDD mutnts, HBsAg espe mutnts, or drug-resistnt mutnts, s this region is essentil for the ssemly nd seretion of suvirl prtiles, nd tolertes only minor struturl hnges. 10 The ssy is stndrdized ginst the World Helth Orgniztion (WHO) Interntionl Stndrd for Heptitis B Virus DNA (NIBSC). 11 Results re reported in Interntionl Units per milliliter (IU/mL) or opies/ml. BIOLOGICAL PRINCIPLES OF THE PROCEDURE The Aott RelTime HBV ssy onsists of three regent kits: Aott RelTime HBV Amplifition Regent Kit Aott RelTime HBV Control Kit Aott RelTime HBV Clirtor Kit The Aott RelTime HBV ssy uses PCR to generte mplified produt from the DNA genome of HBV in linil speimens. A DNA sequene tht is unrelted to the HBV trget sequene is introdued into eh speimen t the eginning of smple preprtion. This unrelted DNA sequene is simultneously mplified y PCR nd serves s n internl ontrol (IC) to demonstrte tht the proess hs proeeded orretly for eh smple. The mount of HBV trget sequene tht is present t eh mplifition yle is mesured through the use of fluoresent-leled oligonuleotide speifilly ound to the mplified produt. The mplifition yle t whih fluoresent signl is deteted y the Aott m2000rt is inversely proportionl to the log of the HBV DNA onentrtion present in the originl smple. Smple Preprtion The purpose of smple preprtion is to extrt nd onentrte nulei id, to mke the trget essile for mplifition, nd to remove potentil inhiitors of mplifition from the extrt. This proess is omplished y the m2000sp, n utomted smple preprtion system designed to use mgneti miroprtile proesses for the purifition of nulei ids from smples. The ssy is suitle for use with oth 0.5 ml nd 0.2 ml smple input volumes. The Aott msmple Preprtion System DNA (4 x Preps) regents lyse the virion, pture the nulei ids, nd wsh the prtiles to remove unound smple omponents. Proteinse K is inluded in the lysis step to digest proteins ssoited with the nulei ids.,13 The ound nulei ids re eluted nd trnsferred to 96-deep well plte. The nulei ids re then redy for mplifition. The IC is introdued into the smple preprtion proedure nd is proessed long with the lirtors, ontrols, nd speimens. Regent Preprtion nd Retion Plte Assemly The Aott m2000sp omines the Aott RelTime HBV mplifition regent omponents (HBV Oligonuleotide Regent, DNA Polymerse, nd Ativtion Regent). The Aott m2000sp dispenses the resulting mster mix to the Aott 96-Well Optil Retion Plte long with liquots of the nulei id smples prepred y the Aott m2000sp. After mnul pplition of the Aott Optil Adhesive Cover, the plte is redy for trnsfer to the Aott m2000rt. Amplifition During the mplifition/detetion retion on the m2000rt instrument, the trget DNA is mplified y the DNA Polymerse in the presene of deoxynuleotide triphosphtes (dntps) nd mgnesium. First, the HBV nd IC primers nnel to their respetive trgets nd re extended y the polymerse. After denturtion step in whih the temperture of the retion is rised ove the melting point of the doule-strnded DNA produt, the newly reted DNA strnd is dentured from the trget DNA. During eh round of therml yling, mplifition produts dissoite to single strnds t high temperture, llowing primer nneling nd extension s the temperture is lowered. Exponentil mplifition of the trget is hieved through repeted yling etween high nd lower tempertures. Amplifition of oth trgets (HBV nd IC) tkes ple simultneously in the sme retion. The trget sequene for the Aott RelTime HBV ssy is in the Surfe gene in the HBV genome. This region is speifi for HBV nd is highly onserved. The primers re designed to hyridize to this region with the fewest possile mismthes mong HBV genotypes A through H. The IC trget sequene is derived from the hydroxypyruvte redutse gene from the pumpkin plnt Cuurit pepo, nd is provided s DNA plsmid in uffer solution. Detetion The presene of HBV mplifition produts is deteted during the extension/nnel step y mesuring the fluoresene of the HBV proe tht inds to the trget during the extension/nnel step. Similrly, the presene of IC mplifition is deteted during the extension/nnel step y mesuring the fluoresene of the IC proe. The HBV nd IC proes re single-strnded DNA oligonuleotides onsisting of proe sequene with fluoresent moiety tht is ovlently linked to the 5' end of the proe nd quenhing moiety tht is ovlently linked to the 3' end of the proe. In the sene of the HBV or IC trget sequenes, proe fluoresene is quenhed. In the presene of HBV or IC trget, the HBV or IC proes speifilly ind to their trget. During the extension/nnel step, the DNA polymerse leves, or nuleolytilly digests, the ound proe s it moves long the templte strnd. This seprtes the fluorophore from the quenher, llowing fluoresent emission nd detetion.
The HBV nd IC proes re eh leled with different fluorophore, thus llowing for simultneous detetion of oth mplified produts t eh yle. The mplifition yle t whih fluoresent signl is deteted y the Aott m2000rt is inversely proportionl to the log of the HBV DNA onentrtion present in the originl smple. PREVENTION OF NUCLEIC ACID CONTAMINATION The possiility of nulei id ontmintion is minimized euse: The Aott RelTime HBV ssy performs PCR mplifition nd oligonuleotide hyridiztion in seled 96-Well Optil Retion Plte. Detetion is rried out utomtilly without the need to open the 96-Well Optil Retion Plte. Aerosol rrier pipette tips re used for ll pipetting. The pipette tips re disrded fter use. Seprte dedited res re used to perform the Aott RelTime HBV ssy. Refer to the SPECIAL PRECAUTIONS setion of this pkge insert. REAGENTS Aott RelTime HBV Amplifition Regent Kit (List No. 2N40-90) 1. Aott RelTime HBV Internl Control (List No. 2G34Y) (4 vils, 0.2 ml per vil) Less thn 0.01% noninfetious linerized DNA plsmid in uffer solution with rrier DNA. Preservtives: 0.085% Sodium zide nd 0.15% ProClin 950. 2. Aott RelTime HBV Amplifition Regent Pk (List No. 2N40) (4 pks, tests/pk) 1 ottle (0.078 ml) DNA Polymerse (5.4 to 5.9 Units/µL) in uffered solution with stilizers. 1 ottle (0.917 ml) HBV Oligonuleotide Regent. Less thn 0.1% syntheti oligonuleotides (4 primers nd 3 proes), nd less thn 0.2% dntps in uffered solution with referene dye. Preservtives: 0.085% Sodium zide nd 0.15% ProClin 950. 1 ottle (0.778 ml) Ativtion Regent. 38 mm mgnesium hloride in uffered solution. Preservtives: 0.085% Sodium zide nd 0.15% ProClin 950. Aott RelTime HBV Control Kit (List No. 2N40-80) 1. Aott RelTime HBV Negtive Control (List No. 2G34Z) (8 vils, 1.3 ml per vil) Negtive humn plsm found to e nonretive y FDA liensed tests for ntiody to HCV, ntiody to HIV-1, ntiody to HIV-2, nd HBsAg. The mteril is lso tested nd found to e negtive y FDA liensed PCR methods for HIV-1 RNA nd HCV RNA. Preservtives: 0.1% ProClin 300 nd 0.15% ProClin 950. 2. Aott RelTime HBV Low Positive Control (List No. 2G34W) (8 vils, 1.3 ml per vil) Het-intivted plsm retive for HBV DNA in negtive humn plsm. Negtive humn plsm found to e nonretive y FDA liensed tests for ntiody to HCV, ntiody to HIV-1, ntiody to HIV-2, nd HBsAg. The mteril is lso tested nd found to e negtive y FDA liensed PCR methods for HIV-1 RNA nd HCV RNA. Preservtives: 0.1% ProClin 300 nd 0.15% ProClin 950. 3. Aott RelTime HBV High Positive Control (List No. 2G34X) (8 vils, 1.3 ml per vil) Het-intivted plsm retive for HBV DNA in negtive humn plsm. Negtive humn plsm found to e nonretive y FDA liensed tests for ntiody to HCV, ntiody to HIV-1, ntiody to HIV-2, nd HBsAg. The mteril is lso tested nd found to e negtive y FDA liensed PCR methods for HIV 1 RNA nd HCV RNA. Preservtives: 0.1% ProClin 300 nd 0.15% ProClin 950. Aott RelTime HBV Clirtor Kit (List No. 2N40-70) 1. Aott RelTime HBV Clirtor A (List No. 2G34A) ( vils, 1.3 ml per vil) Less thn 0.01% noninfetious linerized HBV DNA plsmid in uffer solution. Preservtives: 0.085% Sodium zide nd 0.15% ProClin 950. 2. Aott RelTime HBV Clirtor B (List No. 2G34B) ( vils, 1.3 ml per vil) Less thn 0.01% noninfetious linerized HBV DNA plsmid in uffer solution. Preservtives: 0.085% Sodium zide nd 0.15% ProClin 950. WARNINGS AND PRECAUTIONS In Vitro Dignosti Medil Devie For In Vitro Dignosti Use Only. The Aott RelTime HBV ssy is not intended for use in the sreening of lood, plsm, or tissue donors for HBV, or to e used s dignosti test to onfirm the presene of HBV infetion. Use only USP Grde 190-200 Proof Ethnol (95-100% Ethnol) to prepre the mwsh2 DNA smple preprtion regent. Do not use ethnol tht ontins denturnts. Sfety Preutions Refer to the Hzrd Setions of the Aott m2000sp Opertions Mnul nd the Aott m2000rt Opertions Mnul for instrutions on sfety preutions. CAUTION: This produt ontins humn soured nd/or potentilly infetious omponents. For speifi listing, refer to the REAGENTS setion of this pkge insert. Negtive humn plsm hs een found to e nonretive y FDA liensed tests for ntiody to HCV, ntiody to HIV-1, ntiody to HIV-2, nd HBsAg. The mteril is lso tested nd found to e negtive y FDA liensed PCR methods for HIV-1 RNA nd HCV RNA. No known test method n offer omplete ssurne tht produts derived from humn soures or intivted miroorgnisms will not trnsmit infetion. These regents nd humn speimens should e hndled s if infetious, using sfe lortory proedures, suh s those outlined in Biosfety in Miroiologil nd Biomedil Lortories, 14 OSHA Stndrd on Bloodorne Pthogens, 15 CLSI Doument M29-A3, 16 nd other pproprite iosfety prties. 17 Therefore, ll humn soured mterils should e onsidered potentilly infetious. These preutions inlude, ut re not limited to, the following: Wer gloves when hndling speimens or regents. Do not pipette y mouth. Do not et, drink, smoke, pply osmetis, or hndle ontt lenses in res where these mterils re hndled. Clen nd disinfet spills of speimens y inluding the use of tueruloidl disinfetnt suh s 1.0% sodium hypohlorite or other suitle disinfetnt. 18,19 Deontminte nd dispose of ll potentilly infetious mterils in ordne with lol, stte, nd federl regultions. 20,21 The Aott RelTime HBV Clirtors nd Controls ontin methylisothizolines s omponents of ProClin nd re lssified per pplile Europen Community (EC) Diretives s: Irritnt (Xi). The following re the pproprite Risk (R) nd Sfety (S) phrses: R43 My use sensitiztion y skin ontt. R36/38 Irritting to eyes nd skin. S Avoid ontt with skin. S25 Avoid ontt with eyes. S35 This mteril nd its ontiner must e disposed of in sfe wy. S37 Wer suitle gloves. S39 Wer suitle eye/fe protetion. S46 If swllowed, seek medil dvie immeditely nd show this ontiner or lel. Speil Preutions Hndling Preutions The Aott RelTime HBV ssy is only for use with humn serum nd plsm speimens tht hve een hndled nd stored in pped tues s desried in the SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE setion. During preprtion of smples, ompline with good lortory prties is essentil to minimize the risk of ross-ontmintion etween smples, nd the indvertent introdution of nuleses into smples during nd fter the extrtion proedure. Proper septi tehnique should lwys e used when working with DNA. Amplifition retions suh s PCR re sensitive to identl introdution of produt from previous mplifition retions. Flse positive results ould our if either the linil speimen or the RelTime regents used in the mplifition step eome ontminted y identl introdution of even few moleules of mplifition produt. Mesures to redue the risk of ontmintion in the lortory inlude physilly seprting the tivities involved in performing PCR in ompline with good lortory prties. Work Ares Use two dedited res within the lortory for performing the Aott RelTime HBV ssy. The Smple Preprtion Are is dedited to proessing smples (speimens, Aott RelTime HBV Controls nd Clirtors) nd to dding proessed smples, ontrols, nd lirtors to the Aott 96-Well Optil Retion Plte. The Aott m2000sp omines the Aott RelTime HBV mplifition regent omponents to rete the mplifition mster mix nd trnsfers liquots of the mster mix to the retion plte. All regents used in the Smple Preprtion Are should remin in this dedited re t ll times. Lortory ots, pipettes, pipette tips, nd vortexers used in the Smple Preprtion Are must remin in this re nd not e moved to the Amplifition Are. Do not ring mplifition produt into the Smple Preprtion Are. The Amplifition Are is dedited to the mplifition nd detetion of mplified produt. Lortory ots nd equipment used in the Amplifition Are must remin in this re nd not e moved to the Smple Preprtion Are. Components ontined within kit re intended to e used together. Do not mix omponents from different kit lots. For exmple, do not use the negtive ontrol from ontrol kit lot X with the positive ontrols from ontrol kit lot Y. Do not use kits or regents eyond expirtion dte. Work res nd instrument pltforms must e onsidered potentil soures of ontmintion. Chnge gloves fter ontt with potentil ontminnts (speimens, elutes, nd/or mplified produt) efore hndling unopened regents, negtive ontrol, positive ontrols, lirtors, or speimens. Refer to the Aott m2000sp Opertions Mnul nd the Aott m2000rt Opertions Mnul for instrument lening proedures. If the Aott m2000sp instrument run is orted, dispose of ll ommodities nd regents ording to the Aott m2000sp Opertions Mnul. If the Aott m2000sp mster mix ddition protool is orted, sel the Aott 96-Well Optil Retion Plte in selle plsti g nd dispose ording to the m2000sp Opertions Mnul, Hzrds setion, long with the gloves used to hndle the plte. If the Aott m2000rt instrument run is interrupted or orted, sel the Aott 96 Well Optil Retion Plte in selle plsti g nd dispose ording to the Aott m2000rt Opertions Mnul long with the gloves used to hndle the plte. Deontminte nd dispose of ll speimens, regents, nd other potentilly iohzrdous mterils in ordne with lol, stte, nd federl regultions. 20,21 All mterils should e hndled in mnner tht minimizes the hne of potentil ontmintion of the work re. Note: Autolving the seled Retion Plte will not eliminte the mplified produt nd my ontriute to the relese of the mplified produt y opening of the sel. The lortory re n eome ontminted with mplified produt if the wste mterils re not refully hndled nd ontined efore nd fter proessing. 2
Aerosol Continment To redue the risk of nulei id ontmintion due to erosols formed during mnul pipetting, erosol rrier pipette tips must e used for ll mnul pipetting. The pipette tips must e used only one time. Clen nd disinfet spills of speimens nd regents s stted in the Aott m2000sp or the Aott m2000rt Opertions Mnuls. Contmintion nd Inhiition The following preutions should e oserved to minimize the risks of DNse ontmintion, ross-ontmintion etween smples, nd inhiition: Wer pproprite personl protetive equipment t ll times. Use powder-free gloves. Chnge gloves fter ontt with potentil ontminnts (speimens, elutes, nd/or mplified produt). To redue the risk of nulei id ontmintion due to erosols formed during mnul pipetting, pipettes with erosol rrier tips must e used for ll pipetting. The length of the tip should e suffiient to prevent ontmintion of the pipette rrel. While pipetting, re should e tken to void touhing the pipette rrel to the inside of the smple tue or ontiner. The use of extended erosol rrier pipette tips is reommended. Chnge erosol rrier pipette tips etween ALL mnul liquid trnsfers. Clen nd disinfet spills of speimens nd regents s stted in the Aott m2000sp nd the Aott m2000rt Opertions Mnuls, Hzrds setion. Reple ny empty or prtilly used 200 µl nd 1000 µl disposle tip trys with full trys efore every run. The Aott msmple Preprtion System DNA (4 x Preps) nd Aott Proteinse K regents re single use only. Use new regent vessels, retion vessels, nd newly opened regents for every new Aott RelTime HBV ssy run. At the end of eh run, disrd ll remining regents from the worktle s stted in the Aott m2000sp Opertions Mnul nd the Aott msmple Preprtion System DNA (4 x Preps) nd Aott Proteinse K pkge inserts. STORAGE INSTRUCTIONS Aott RelTime HBV Amplifition Regent Kit (List No. 2N40-90) The Aott RelTime HBV Amplifition Regent Pk nd Internl Control vils must e stored t -10 C or older when not in use. Cre must e tken to seprte the Aott RelTime HBV Amplifition Regent Pk tht is in use from diret ontt with smples, lirtors, nd ontrols. Aott RelTime HBV Control Kit (List No. 2N40-80) The Aott RelTime HBV Negtive nd Positive Controls must e stored t -10 C or older. Aott RelTime HBV Clirtor Kit (List No. 2N40-70) The Aott RelTime HBV Clirtor A nd Clirtor B must e stored t -10 C or older. SHIPPING CONDITIONS Aott RelTime HBV Amplifition Regent Kit: Ship on dry ie. Aott RelTime HBV Control Kit: Ship on dry ie. Aott RelTime HBV Clirtor Kit: Ship on dry ie. INDICATION OF INSTABILITY OR DETERIORATION OF REAGENTS When positive or negtive ontrol vlue is out of the expeted rnge, it my indite deteriortion of the regents. Assoited test results re invlid nd smples must e retested. Assy relirtion my e neessry. Refer to the QUALITY CONTROL PROCEDURES: Assy Clirtion setion of this pkge insert for detils. If you reeive regents, lirtors, or ontrols tht re in ondition ontrry to lel reommendtion, or tht re dmged, ontt Aott Moleulr Tehnil Servies. SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE Speimen Colletion nd Storge Humn serum nd plsm (EDTA) speimens my e used with the Aott RelTime HBV ssy. Follow the mnufturer s instrutions for proessing olletion tues. Freshly drwn speimens (whole lood) my e held t 2 to 30 C for up to 6 hours prior to entrifugtion. After entrifugtion, remove serum or plsm from ells. Serum or plsm speimens my e stored: At 15 to 30 C for up to hours At 2 to 8 C for up to 3 dys At -20 C or older for longer term Multiple freeze-thw yles should e voided. If frozen, thw speimens t 15 to 30 C or t 2 to 8 C. One thwed, if speimens re not eing proessed immeditely, they n e stored t 2 to 8 C for up to 6 hours. Speimen Trnsport Ship speimens frozen on dry ie. For domesti nd interntionl shipments, speimens should e pkged nd leled in ompline with pplile stte, federl, nd interntionl regultions overing the trnsport of linil, dignosti, or iologil speimens. INSTRUMENT PROCEDURE The Aott RelTime HBV pplition file(s) must e instlled on the Aott m2000sp nd Aott m2000rt instruments from the Aott RelTime HBV m2000 System Comined Applition CD-ROM prior to performing the ssy. For detiled informtion on pplition file instlltion, refer to the Aott m2000sp nd m2000rt Opertions Mnuls, Operting Instrutions setion. ABBOTT REALTIME HBV ASSAY PROCEDURE Mterils Provided Aott RelTime HBV Amplifition Regent Kit (List No. 2N40-90) Mterils Required But Not Provided Aott RelTime HBV Control Kit (List No. 2N40-80) Aott RelTime HBV Clirtor Kit (List No. 2N40-70) Smple Preprtion Are Aott m2000sp Aott msmple Preprtion System DNA (4 x Preps) (List No. 06K-) Aott Proteinse K (List No. 3L78-60) Aott RelTime HBV m2000 System Comined Applition CD-ROM (List No. 2N43) Smple Rks 5 ml Retion Vessels 200 ml Regent Vessels Mster Mix Vil Aott 96-Well Optil Retion Plte Aott 96-Deep Well Plte Aott Splsh-Free Support Bse Aott Optil Adhesive Cover Aott Adhesive Cover Applitor Round-ottom.5 x 75 mm Smple Tues Vortex Mixer 50 ml Polypropylene Centrifuge Tues Centrifuge ple of 2,000g Clirted Preision Pipettes ple of delivering 10 µl-1000 µl 20 µl-1000 µl Aerosol Brrier Pipette Tips for preision pipettes Serologil Pipettes Grduted Cylinder, 100 ml USP Grde 190-200 Proof Ethnol (95-100% Ethnol). Do not use ethnol tht ontins denturnts. Moleulr Biology Grde Wter 1.7 ml Moleulr Biology Grde Miroentrifuge Tues (Dot Sientifi, In. or equivlent)* Cotton Tip Applitors (Puritn or Equivlent)* *Note: These items re used in the proedure for Monitoring the Lortory for the Presene of Amplifition Produt. Refer to the QUALITY CONTROL PROCEDURES setion of this pkge insert. Amplifition Are Aott m2000rt Aott RelTime HBV m2000 System Comined Applition CD-ROM (List No. 2N43) Aott m2000rt Optil Clirtion Kit (List No. 4J71-93) Other Mterils Biologil sfety inet pproved for working with infetious mterils. Selle plsti gs Proedurl Preutions Red the instrutions in this pkge insert refully efore proessing smples. The Aott RelTime HBV Clirtors, Internl Control, Negtive Control, nd Low nd High Positive Control vils re intended for single-use only nd should e disrded fter use. Smple tues should e inspeted for ir ules. If found, remove them with sterile pipette tip. Regent ules my interfere with proper detetion of regent levels in the regent vessel, using insuffiient regent spirtion, whih ould impt results. Cution should e tken to void ross-ontmintion etween smples y using new sterile pipette tip for eh tue. Use erosol rrier pipette tips or disposle pipettes only one time when pipetting speimens, ontrols, lirtors, or Amplifition Regents. To prevent ontmintion to the pipette rrel while pipetting, re should e tken to void touhing the pipette rrel to the inside of the smple tue or ontiner. The use of extended erosol rrier pipette tips is reommended. Monitoring proedures for the presene of mplifition produt n e found in the QUALITY CONTROL PROCEDURES setion in this pkge insert. To redue the risk of nulei id ontmintion, len nd disinfet spills of speimens y inluding the use of tueruloidl disinfetnt suh s 1.0% sodium hypohlorite or other suitle disinfetnt. A lirtion urve must e estlished efore speimens re tested. The use of the Aott RelTime HBV Clirtors nd Controls is integrl to the performne of the Aott RelTime HBV ssy. Refer to the QUALITY CONTROL PROCEDURES setion of this pkge insert for detils. ASSAY PROTOCOL Smple Preprtion Are All speimen storge nd preprtion must tke ple in the dedited Smple Preprtion Are. Refer to the Hndling Preutions setion of this pkge insert for instrutions efore prepring smples. 3
For detiled desription of how to operte the Aott m2000sp instrument nd Aott m2000rt instrument, refer to the Aott m2000sp nd m2000rt Opertions Mnuls, Operting Instrutions setion. Lortory personnel must e trined to operte the Aott m2000sp nd m2000rt instruments. The opertor must hve thorough knowledge of the pplitions run on the instruments nd must follow good lortory prties. 1. A mximum of smples n e proessed in eh run. A negtive ontrol, low positive ontrol, nd high positive ontrol must e inluded in eh run, therefore llowing mximum of 45 speimens to e proessed per run. NOTE: If performing run of more thn smples, empty the solid wste ontiner efore the run nd reple with new iohzrd g if ny wste is present. Chek smple volume. The Aott RelTime HBV ssy minimum smple volume nd ssoited rk requirements on the Aott m2000sp re: Aott RelTime HBV Minimum Smple Volume Assy Applition Rk Tue Dimeter* 0.2 ml 0.5 ml 13 mm 11.5 mm - 14.0 mm 0.4 ml - 0.8 ml 0.7 ml - 1.2 ml 16 mm 14.5 mm - 16.0 mm 0.4 ml - 1.0 ml 0.8 ml - 1.4 ml * Refers to the smple tue outer dimeter. Minimum smple volume vries with tue geometry nd size. Refer to the m2000sp Opertions Mnul nd QUICK REFERENCE GUIDE FOR SAMPLE TUBE SIZES AND VOLUMES for reommended smple input volume. If frozen, thw speimens t 15 to 30 C or t 2 to 8 C. One thwed, if speimens re not eing proessed immeditely, store t 2 to 8 C for up to 6 hours. Before use, vortex speimens three times for 2 to 3 seonds. Ensure tht ules or fom re not reted. If found, remove them with new sterile pipette tip for eh tue. Speimens showing prtiulte mtter or turidity should e lrified y entrifugtion t 2,000g for 5 minutes prior to testing. Aliquot eh speimen into len tues or vils if neessry. Refer to the Aott m2000sp Opertions Mnul for tue sizes. Avoid touhing the inside of the p when opening tues. 2. Thw ssy ontrols nd Internl Control (IC) t 15 to 30 C or t 2 to 8 C. Thw lirtors t 15 to 30 C or t 2 to 8 C only if performing lirtion run; see QUALITY CONTROL PROCEDURES setion of this pkge insert. One thwed, if lirtors, ontrols, nd IC re not eing proessed immeditely, store t 2 to 8 C for up to hours. Vortex eh ssy lirtor nd eh ontrol three times for 2 to 3 seonds efore use. Ensure tht ules or foming re not reted. If found, remove them with new sterile pipette tip for eh tue. Ensure tht the ontents of the vils re t the ottom fter vortexing y tpping the vils on the enh to ring liquid to the ottom of the vils. 3. Thw mplifition regents t 15 to 30 C or t 2 to 8 C until required for the mplifition mster mix proedure. This step n e initited efore ompletion of the smple preprtion proedure. Note: Do not vortex the Amplifition Regent Pk. One thwed, the mplifition regents n e stored t 2 to 8 C for up to hours if not used immeditely. 4. Open the Aott Proteinse K regent pk. Add 17.15 ml of Moleulr Biology Grde wter to 50 ml polypropylene entrifuge tue. Pipet 2.45 ml of Proteinse K into the ontiner of wter. Mix y gentle inversion 10 to 15 times. Trnsfer the entire ontents to regent vessel leled with the Proteinse K rode lel. Ple the regent vessel in regent rrier #1 lotion 2. NOTE: The following steps 5 through 9 pertin to the use of the msmple Preprtion System DNA kit (List No. 06K-). NOTE: Use one ottle of Proteinse K solution, one set of the msmple Preprtion System DNA regents, one vil of IC, nd one RelTime HBV Amplifition Regent Pk to support up to retions. Use seond set of regents to support 25 to retions, with the exeption of the mmiroprtiles DNA. One ottle of mmiroprtiles DNA will support up to retions. Do not use more thn one ottle of mmiroprtiles DNA. 5. Open the Aott msmple Preprtion pk. If rystls re oserved in ny of the regent ottles upon opening, llow the regents to equilirte t room temperture until the rystls dispper. Do not use the regents until the rystls hve dissolved. 6. Prepre the mwsh2 DNA y dding 70 ml of USP Grde 190-200 Proof Ethnol (95-100% Ethnol) to the mwsh2 DNA ottle s desried in the Aott msmple Preprtion System DNA produt informtion. Do not use ethnol tht ontins denturnts. 7. Vortex the IC vil(s) three times for 2 to 3 seonds efore use. 8. Using lirted preision PIPETTE DEDICATED FOR INTERNAL CONTROL USE ONLY, dd 100 µl of IC to ottle of mlysis Buffer. Mix y gently inverting the ontiner 5 to 10 times to minimize foming. 9. Gently invert the Aott msmple Preprtion ottles to ensure homogeneous solution nd pour the ontents into the pproprite regent vessels per the Aott m2000sp Opertions Mnul, Operting Instrutions. 10. Ple the low nd high positive ontrols, the negtive ontrol, the lirtors (if pplile), nd the ptient speimens into the m2000sp smple rk. 4 11. Ple the 5 ml Retion Vessels into the m2000sp 1 ml susystem rrier.. Lod the rrier rks ontining the Aott msmple Preprtion regents nd Proteinse K, nd the Aott 96-Deep Well Plte, on the Aott m2000sp worktle s desried in the Aott m2000sp Opertions Mnul, Operting Instrutions. 13. From the Run Smple Extrtion sreen, selet the pproprite pplition file orresponding to the smple volume eing tested. Initite the smple extrtion protool s desried in the m2000sp Opertions Mnul, Operting Instrutions. Enter lirtor (needed if lirtion urve hs not een stored on the m2000rt) nd ontrol lot speifi vlues in the Smple Extrtion: Assy Detils sreen. Lot speifi vlues re speified in eh Aott RelTime HBV Clirtor nd Control Kit rd. NOTE: Verify the vlues entered mth the vlues on the kit rds. The Aott m2000sp Mster Mix Addition protool (step 14) must e initited within 60 minutes fter ompletion of the Smple Extrtion protool. NOTE: Chnge gloves efore hndling the mplifition regents. 14. Lod the mplifition regents, the mster mix vil, nd the 96-well Optil Retion Plte on the m2000sp worktle fter smple preprtion is ompleted. Eh Amplifition Regent Pk supports up to retions. Ensure the mplifition regents re thoroughly thwed efore use. Prior to opening the mplifition regents, ensure tht the ontents re t the ottom of the vils y tpping the vils in n upright position on the enh. Remove nd disrd the mplifition vil ps. A seond Amplifition Regent Pk is required if performing 25 to retions. 15. Selet the pproprite deep well plte from the Run Mster Mix Addition sreen tht mthes the orresponding smple preprtion extrtion. Initite the Aott m2000sp Mster Mix Addition protool. Follow the instrutions s desried in the Aott m2000sp Opertions Mnul, Operting Instrutions setion. The m2000rt protool (step 20) must e strted within 60 minutes of the ompletion of the Mster Mix Addition protool (step 15). Amplifition Are 16. Swith on nd initilize the Aott m2000rt in the Amplifition Are. The Aott m2000rt requires 15 minutes to wrm up. NOTE: Remove gloves efore returning to the Smple Preprtion Are. Smple Preprtion Are 17. Sel the Aott 96-Well Optil Retion Plte fter the Aott m2000sp instrument hs ompleted ddition of smples nd mster mix ording to the Aott m2000sp Opertions Mnul, Operting Instrutions setion. NOTE: Sine mximum of smples n e proessed in eh run, the 96-Well Optil Retion Plte will ontin empty wells. 18. Ple the 96-Well Optil Retion Plte into the Splsh-Free Support Bse for trnsfer to the Aott m2000rt instrument. 19. Export the ompleted 96-Well Optil Retion Plte results to CD or Network Drive. Amplifition Are 20. Ple the Aott 96-Well Optil Retion Plte in the Aott m2000rt instrument. Import the m2000sp test order vi CD or Network Drive per the Import Order instrutions in the Aott m2000rt Opertions Mnul, Operting Instrutions setion. POST PROCESSING PROCEDURES 21. Remove the Aott 96-Deep Well Plte from the worktle nd dispose ording to the Aott m2000sp Opertions Mnul. 22. Ple the Aott 96-Well Optil Retion Plte in selle plsti g nd dispose ording to the Aott m2000rt Opertions Mnul, long with the gloves used to hndle the plte. 23. Clen the Splsh Free Support Bse efore next use, ording to the Aott m2000rt Opertions Mnul. QUALITY CONTROL PROCEDURES Aott m2000rt Optil Clirtion Refer to the Clirtion Proedures setion in the Aott m2000rt Opertions Mnul for detiled desription of how to perform n Aott m2000rt Optil Clirtion. Optil lirtion of the Aott m2000rt instrument is required for the urte mesurement nd disrimintion of dye fluoresene during the Aott RelTime HBV ssy. The following Aott m2000rt Optil Clirtion Pltes re used to lirte the Aott m2000rt instrument for the Aott RelTime HBV ssy: FAM Plte (Croxyfluoresein) ROX Plte (Croxy-X-rhodmine) VIC Plte (Proprietry dye) Assy Clirtion A lirtion urve is required to quntitte HBV DNA in the speimens nd ontrols. Two ssy lirtors re run in replites of three to generte lirtion urve (HBV onentrtion [log IU/mL] versus the threshold yle [Ct] t whih retive level of fluoresent signl is deteted). The lot speifi vlues for Clirtor A nd Clirtor B re speified on eh Aott RelTime HBV Clirtor Kit Crd nd must e entered into the ssy test order when run is performed. The lirtion urve slope nd interept re lulted nd stored on the instrument. The onentrtion of HBV DNA in smple is lulted from the lirtion urve. Results re utomtilly reported on the m2000rt worksttion.
The Low nd High Positive Controls nd Negtive Control must e inluded in the lirtion run. Follow the proedure for smple extrtion, regent ddition, mplifition nd detetion protools s stted in the Aott m2000sp Opertions Mnul nd the m2000rt Opertions Mnul. Ensure tht ssy ontrol vlues oserved in the finl report re within the rnges speified on the Aott RelTime HBV Control Kit Crd. One n Aott RelTime HBV lirtion is epted nd stored, it my e used for 6 months. During this time, ll susequent smples my e tested without further lirtion unless: An Aott RelTime HBV Amplifition Regent Kit with new lot numer is used. An Aott msmple Preprtion System DNA (4 x Preps) with new lot numer is used. An Aott RelTime HBV pplition speifition file for different smple volume is used. An updted version of the Aott RelTime HBV pplition speifition file is instlled. Detetion of Inhiition An IC threshold yle [Ct] ssy vlidity prmeter is estlished during lirtion run. Prior to smple preprtion, defined, onsistent quntity of the IC is introdued into the lysis uffer, whih is then used during the proessing of eh speimen, lirtor, nd ontrol, nd mesured on the m2000rt instrument to demonstrte proper smple proessing nd ssy vlidity. The IC is omposed of DNA sequene unrelted to the HBV DNA sequene. The medin mplifition yle t whih the IC trget sequene fluoresent signl is deteted in lirtion smples estlishes the IC Ct vlidity rnge to e met y ll susequent proessed speimens using tht lirtion urve. An error is displyed when speimen or ontrol fils to meet this speifition. Refer to the Aott m2000rt Opertions Mnul for n explntion of the orretive tions for the error ode. Speimens whose IC Ct vlue flls outside of the estlished rnge must e retested strting with smple preprtion. Negtive nd Positive Controls A negtive ontrol, low positive ontrol, nd high positive ontrol re inluded in eh run to evlute run vlidity. The lot speifi vlues for the low positive ontrol nd high positive ontrol re speified on eh Aott RelTime HBV Control Kit Crd nd must e entered into the test order when run is performed. An error is displyed when ontrol result is out of rnge. Refer to the Aott m2000rt Opertions Mnul for n explntion of the orretive tions for the error ode. If negtive or positive ontrols re out of rnge, ll of the speimens nd ontrols from tht run must e reproessed, eginning with smple preprtion. The presene of HBV must not e deteted in the negtive ontrol. HBV deteted in the negtive ontrol is inditive of ontmintion y other smples or y mplified produt introdued during smple preprtion or during preprtion of the Aott 96- Well Optil Retion Plte. To void ontmintion, len the Aott m2000sp nd m2000rt instruments nd repet the smple proessing for ontrols nd speimens following the Proedurl Preutions. If negtive ontrols re persistently retive, ontt your Are Customer Support representtive. Monitoring the Lortory for the Presene of Amplifition Produt It is reommended tht this test e done t lest one month to monitor lortory surfes nd equipment for ontmintion y mplifition produt. It is very importnt to test ll res tht my hve een exposed to proessed speimens nd ontrols, lirtors, nd/or mplifition produt. This inludes routinely hndled ojets suh s pipettes, the Aott m2000sp nd m2000rt funtion keys, lortory enh surfes, miroentrifuges, nd entrifuge dptors. 1. Add 0.8 ml Moleulr Biology Grde wter to 1.7 ml DNse-free miroentrifuge tue. 2. Sturte the otton tip of n pplitor (Puritn or equivlent) in the Moleulr Biology Grde wter from the miroentrifuge tue. 3. Using the sturted otton tip of the pplitor, wipe the re to e monitored using sweeping motion. Ple the pplitor into the miroentrifuge tue. 4. Swirl the otton tip in Moleulr Biology Grde wter 10 times, nd then press the pplitor long the inside of the tue so tht the liquid drins k into the solution t the ottom of the miroentrifuge tue. Disrd the pplitor. 5. Pipette 0.5 ml of the mwsh 1 uffer to len tue using the pipette dedited for Internl Control use. 6. Add 20 µl of the mwsh 1 uffer to eh miroentrifuge tue. 7. Cp the miroentrifuge tue. 8. Test this smple ording to the ssy proedure setion of this pkge insert. 9. Trnsfer liquid from miroentrifuge tue to 5 ml Retion Vessel. 10. Bring the volume to 1.5 ml with Moleulr Biology Grde wter. 11. The presene of ontmintion is indited y the detetion of HBV in the sw smples.. If HBV is deteted on equipment, follow the lening nd deontminting guidelines given in tht equipment s opertions mnul. If HBV is deteted on surfes, len the ontminted res with 1.0% (v/v) sodium hypohlorite solution, followed y 70% ethnol or wter. Note: Chlorine solutions my pit equipment nd metl. Use suffiient mounts or repeted pplitions of 70% ethnol or wter until hlorine residue is no longer visile. 13. Repet testing of the ontminted re y following Steps 1 through 10. RESULTS Clultion The onentrtion of HBV DNA in smple or ontrol is lulted from either stored lirtion urve, or lirtion urve reted y lirtors within lirtion or smple run. The Aott m2000rt instrument utomtilly reports the results on the m2000rt worksttion. Assy results re reported in IU/mL or log IU/mL. Results n lso e reported in opies/ml or log opies/ml using onversion ftor of 3.41 (1 IU = 3.41 opies). Note: The ssy is lirted to the WHO Interntionl HBV DNA Stndrd. The 3.41 onversion ftor is sed on n verge onversion ftor ross the ssy dynmi rnge. The following tle represents the potentil m2000rt outputs tht n e oserved y the user. Interprettion of Results Smple Volume Result Interprettion 0.5 ml Not Deteted Trget not deteted < 1.00 Log IU/mL Deteted 1.00 to 9.00 Log IU/mL d > 9.00 Log IU/mL > ULQ e 0.2 ml Not Deteted Trget not deteted < 1.18 Log IU/mL Deteted 1.18 to 9.00 Log IU/mL d > 9.00 Log IU/mL > ULQ e 10 IU/mL 15 IU/mL Below LLQ (lower limit of quntittion or LLoQ); HBV DNA is not quntifile. d Clulted results re within ssy liner rnge. If lulted result is otined, the Interprettion field is left lnk. e > ULQ ove upper limit of quntittion or ULoQ; if IU/mL results re ove the liner rnge of the ssy, results re reported s >1,000,000,000 IU/mL HBV DNA. If negtive or positive ontrols re out of rnge, ll of the speimens nd ontrols from tht run must e reproessed, eginning with smple preprtion. If quntittive results re desired for those speimens reported s > ULQ (ULoQ), the originl speimen should e diluted 1:50 with HBV-negtive humn plsm or serum (onsistent with the mtrix of the originl speimen), nd the test repeted. Multiply the reported result y the dilution ftor of 50 to otin the quntittive result. Exmple lultions re provided elow. Exmple result for diluted speimen Clulte 10 x (x = result in log unit); round to whole numer Multiply whole numer result y 50 (dilution ftor) to otin result orreted for dilution Clulte log (y) (y = whole numer result) 7.59 log IU/mL 38,904,515 IU/mL 1,945,225,750 IU/mL 9.29 log IU/mL 63,095,734 IU/mL Not pplile 3,154,786,700 IU/mL Not pplile 8.80 log opies/ml 630,957,345 opies/ml 49,905,5 opies/ml 31,547,867,250 opies/ml Not pplile 2,495,262,250 opies/ml 10.50 log opies/ml Not pplile LIMITATIONS OF THE PROCEDURE FOR IN VITRO DIAGNOSTIC USE ONLY. Optiml performne of this test requires pproprite speimen olletion, storge, nd trnsport to the test site (refer to the SPECIMEN COLLECTION, STORAGE, AND TRANSPORT TO THE TEST SITE setion of this pkge insert). Humn serum nd plsm (EDTA) my e used with the Aott RelTime HBV ssy. The use of other ntiogulnts hs not een vlidted for use with the Aott RelTime HBV ssy. The use of speimens olleted in serum tues tht ontin Z-lot tivtor, or similr types of rpid lot tivtor, my use inhiited results in the RelTime HBV ssy. Therefore, serum olletion tues ontining Z-lot tivtor or similr rpid lot tivtors should not e used. The instruments nd ssy proedures redue the risk of ontmintion y mplifition produt. However, nulei id ontmintion from the lirtors, positive ontrols, or speimens must e ontrolled y good lortory prtie nd reful dherene to the proedures speified in this pkge insert. In rre ses, very low level positive result my our from ross ontmintion during proessing of n extremely high opy numer djent speimen. Crryover rtes in representtive studies rnged from 0% to 2%. Per tretment guidelines, 1 log inrese is needed in order to impt ptient mngement. 22,23 In ddition, tretment guidelines require two onseutive elevted mesurements to our efore hnging ptient mngement. A single Entevir muttion (rta97v) ours within the reverse primer. Of ll known resistne muttions, it is the only one tht ours within ny Aott RelTime HBV primer or proe sequene. Softwre simultion predits tht this muttion (rta97v) would not e expeted to interfere with ssy results when using RelTime HBV ssy onditions. A speimen with result of Not Deteted nnot e presumed to e negtive for HBV DNA. 5
Preision ws estlished with HBV s A nd C only. Drug interferene ws evluted using plsm mtrix, nd ws not evluted in serum. The listed drugs were tested in pools, nd individul drug effets were not ssessed. The interferene studies were performed with n HBV DNA onentrtion of 2,933 IU/mL (3.47 log IU/mL). Potentil interferene on HBV DNA onentrtions lose to the ssy LLQ (LLoQ) ws not ssessed. Some of the ross-retivity studies were performed with nulei ids (DNA nd RNA) only. For further detil, refer to the SPECIFIC PERFORMANCE CHARACTERISTICS setion of this pkge insert. Results from the Aott RelTime HBV ssy should e interpreted in onjuntion with other linil nd lortory findings. SPECIFIC PERFORMANCE CHARACTERISTICS The following performne hrteristis were determined using the RelTime HBV ssy with the 0.5 ml smple preprtion proedure, unless otherwise speified. WHO STANDARDIZATION Figure 1 demonstrtes the omprison of Aott RelTime HBV Assy Clirtors to the WHO Interntionl HBV DNA Stndrd. Aott RelTime HBV Clirtors tre to the World Helth Orgniztion (WHO) Interntionl Stndrd for Heptitis B Virus DNA (NIBSC) eh time lot is mnuftured. Eh lot of lirtor is speifilly ssigned quntittion vlue through testing with HBV Primry Clirtors, whih re diretly tested ginst the WHO stndrd. The lot-speifi quntittion vlues for eh HBV lirtor re entered into the m2000rt softwre when run is eing performed. The evlution ws onduted with the WHO 1 st Interntionl HBV DNA Stndrd, nd one lot of HBV Clirtors, nd ws performed on one run. The WHO stndrd ws reonstituted to onentrtion of 1 x 10 5 IU/mL nd then diluted to 1 x 10 4, 1 x 10 3, nd 1 x 10 2 IU/mL in negtive humn plsm. The highest ssy lirtor, Clirtor B, whih is lot-ssigned t 6.42 log IU/mL, ws diluted to 1 x 10 5,1 x 10 4, 1 x 10 3, nd 1 x 10 2 IU/mL in Tris-EDTA (TE) uffer. The dt for Clirtor B nd its dilution series re presented in omprison to the WHO stndrd dilution series in Figure 1. The results indite tht the ssy stndrdiztion proess provides quntittion vlues for the RelTime HBV Clirtors nd the WHO stndrd tht re similr to the expeted vlues, with devition of not more thn 0.33 log IU/mL. The mximum devition ws otined t the ssy ULQ (ULoQ). Figure 1 Comprison of WHO 1 st Interntionl HBV DNA Stndrd with Aott RelTime HBV Clirtors LINEAR RANGE The ULQ (ULoQ) for the Aott RelTime HBV ssy is 10 9 IU/mL (9.00 log IU/mL) nd the LLQ (LLoQ) is equivlent to the LoD, whih is 10 IU/mL (1.00 log IU/mL) for the 0.5 ml smple preprtion protool nd 15 IU/mL (1.18 log IU/mL) for the 0.2 ml smple preprtion protool. In one study, 13-memer pnel prepred y diluting n HBV-positive speimen trgeted from 9.13 log IU/mL to 0.29 log IU/mL in HBV negtive humn plsm ws tested nd evluted in ordne with methods defined in the CLSI EP6-A, 25 using the 0.5 ml smple preprtion protool. The Aott RelTime HBV ssy ws shown to e liner in plsm ross the rnge of HBV DNA onentrtions tested (shown in Figure 2) with devition from linerity of not more thn 0.20 log IU/mL. In seond study, one pnel onsisting of A nd one pnel onsisting of C were tested. The two 10-memer pnels were prepred y diluting to onentrtions trgeted from 1.27 log IU/mL to 8.47 log IU/mL for A nd 1.59 log IU/mL to 8.79 log IU/mL for C. The two pnels were prepred with high opy HBV-positive speimens diluted in HBV serologilly-negtive humn plsm. Lest squres liner regression nlysis ws performed for s A nd C seprtely. Anlysis for A is shown in Figure 3 nd nlysis for C is shown in Figure 4. The Aott RelTime HBV ssy ws shown to e liner in plsm ross the rnge of HBV DNA onentrtions tested for HBV A nd HBV C. Aott Reltime HBV (Log IU/mL) Figure 2 Aott RelTime HBV Liner Rnge Lest Squres Liner Regression Anlysis y = 0.99x + 0.01 r = 0.999 n = 77 Trget Conentrtion (Log IU/mL) Figure 3 Aott RelTime HBV Liner Rnge Lest Squres Liner Regression Anlysis A 9 8 y = 0.99x - 0.10 r = 0.998 n = 80 7 Aott Reltime HBV (Log IU/mL) 6 5 4 3 2 1 0 0 1 2 3 4 5 6 7 8 9 Trget Conentrtion (Log IU/mL) 6
9 8 Figure 4 Aott RelTime HBV Liner Rnge Lest Squres Liner Regression Anlysis C y = 0.99x - 0.09 r = 0.999 n = 80 LINEARITY OF ASSAY BY HBV GENOTYPES The ility of the RelTime HBV ssy to detet nd quntitte HBV genotypes ws evluted through linerity studies y diluting eight speimens, one of eh genotype A through H, to trget onentrtions of 4.47 log IU/mL, 3.47 log IU/mL, 2.47 log IU/mL, nd 1.17 log IU/mL in HBV serologilly negtive humn plsm. Three replites were tested t eh onentrtion for eh genotype, using the 0.5 ml smple preprtion protool. Dt of the studies demonstrted tht RelTime HBV ssy is ple to quntitte different HBV genotypes ross liner rnge with devition of not more thn 0.51 log IU/mL. The results re summrized in Tle 3 nd Figure 5. Aott Reltime HBV (Log IU/mL) 7 6 5 4 3 2 1 0 0 1 2 3 4 Trget Conentrtion (Log IU/mL) 5 6 7 8 9 Tle 3 Aott RelTime HBV Linerity of Assy y HBV s Liner Eqution from Linerity Study Mximum Differene Between A nd Corresponding (Log IU/mL) A y = 0.95x + 0.19 n/ B y = 0.89x + 0.40 0.35 C y = 0.93x + 0.32 0.11 D y = 0.89x + 0.43 0.32 E y = 0.86x + 0.58 0.44 F y = 0.90x + 0.42 0.23 G y = 0.85x + 0.61 0.51 H y = 0.86x + 0.60 0.42 The mximum differene ws otined t the ssy ULQ (ULoQ) or LLQ (LLoQ). Limit of Quntittion The totl nlytil error (TAE) ws lulted using estimtes determined from the reproduiility studies tht were onduted t three sites: two externl sites nd one internl site. s A nd C were tested t oth smple volumes nd in oth plsm nd serum. Presented in Tle 1 re the TAE estimtes for the plsm pnel memers tht hd n oserved onentrtion t or ner the ssy limit of detetion, for eh smple input volume. Presented in Tle 2 re the TAE estimtes for the serum pnel study. TAE ws estimted y two different methods (see tle footnotes). These studies demonstrted tht the Aott RelTime HBV ssy n determine with n eptle level of ury the onentrtion of HBV DNA in EDTA plsm nd serum t onentrtions of 10 IU/mL (1.00 log IU/mL) for the 0.5 ml smple protool volume nd 15 IU/mL (1.18 log IU/mL) for the 0.2 ml smple protool volume. At these onentrtions, the differene etween two mesurements of more thn 1.00 log IU/mL is sttistilly signifint. Figure 5 Aott RelTime HBV Linerity of Assy y HBV s (Dilutionl Linerity) Smple Volume (ml) Tle 1 Aott RelTime HBV Totl Anlytil Error (TAE) Estimtes (Plsm) (Log IU/mL) HBV (Pnel Memer) TAE Asolute TAE d Bis + SQRT (2) (2 x SD) x 2 x SD Expeted Oserved Asolute n Con. Con. Bis SD 0.5 110 A (5) 1.04 0.90 0.14 0.32 0.78 0.91 0.5 119 C (10) 1.14 1.13 0.01 0.29 0.59 0.82 0.2 37 A (5) 1.04 1.03 0.01 0.40 0.81 1.13 0.2 46 C (10) 1.14 1. 0.10 0.30 0.70 0.85 Pnel Memer is elow the ssy LoD (1.00 log IU/mL for 0.5 ml nd 1.18 log IU/mL for 0.2 ml). TAE is provided for informtion only. SD = Within-run omponent vriility + Between-run omponent vriility. Per setion 5.1 of EP17-A CLSI guideline. 26 d Bsed on differene etween two mesurements pproh. Smple Volume (ml) Tle 2 Aott RelTime HBV Totl Anlytil Error (TAE) Estimtes (Serum) (Log IU/mL) HBV (Pnel Memer) TAE Asolute Bis + (2 x SD) TAE d SQRT (2) x 2 x SD Expeted Oserved Asolute n Con. Con. Bis SD 0.5 88 A (6) 1.36 1.04 0.32 0.20 0.72 0.57 0.5 90 C () 1. 1.29 0.19 0.20 0.59 0.57 0.5 88 C (13) 1.27 0.95 0.32 0.25 0.82 0.71 0.2 88 A (5) 1.56 1.10 0.46 0. 0.94 0.68 0.2 89 C () 1. 1.14 0.34 0. 0.82 0.68 Pnel Memer is elow the ssy LoD (1.00 log IU/mL for 0.5 ml nd 1.18 log IU/mL for 0.2 ml). TAE is provided for informtion only. SD = Within-run omponent vriility + Between-run omponent vriility. Per setion 5.1 of EP17-A CLSI guideline. 26 d Bsed on differene etween two mesurements pproh. WITHIN-LABORATORY PRECISION: LOT-TO-LOT The preision of the ssy ws evluted using n eight-memer pnel. Pnel memers 2, 3, 5, nd 7 were prepred y diluting high opy HBV ptient smple in HBV serologilly negtive humn serum. Pnel memers 1, 4, 6, nd 8 were prepred y diluting the sme high opy HBV ptient smple into HBV serologilly negtive humn plsm. A totl of three regent lots were used nd eh lot ws ssigned n m2000sp nd m2000rt instrument pir. A totl of 45 replites were tested for eh pnel memer ross the three pirs of m2000sp nd m2000rt instruments. One run ws performed per dy on eh instrument pir for five dys for totl of 15 runs. Pnel memers 1 through 8 were run in replites of three. The onentrtion levels trgeted for the preision pnels spnned the liner quntittion rnge of the ssy. The 0.5 ml smple preprtion protool ws used. The etween-lot/ instrument omponent of preision ws less or equl to 0.14 log IU/mL. The results re summrized in Tle 4. 7
Tle 4 Aott RelTime HBV Within-Lortory Preision for the 0.5 ml Smple Preprtion Protool Within-Run Component SD Between-Run Component SD Between-Lot/ Instrument Component SD Pnel Speimen Type n Men Con. (Log IU/mL) Totl SD, 1 P 45 1.41 0.19 0.00 0.08 0.21 2 S 45 2.32 0.07 0.04 0.07 0.11 3 S 45 3. 0.06 0.05 0.08 0.11 4 P 45 4.38 0.06 0.06 0.08 0. 5 S 45 5.47 0.09 0.00 0.10 0.13 6 P 45 6.38 0.06 0.07 0.11 0.15 7 S 45 7.54 0.05 0.08 0.14 0.17 8 P 45 8.44 0.04 0.05 0.13 0.14 P = Plsm; S = Serum Stndrd devitions (SD) re in log IU/mL. Totl preision inludes within-run, etween-run nd etween-lot/instrument omponents of preision. WITHIN-LABORATORY PRECISION: OPERATOR-TO-OPERATOR The within-run, etween-run, nd etween-tehniin (opertor) preision of the Aott RelTime HBV ssy ws evluted y testing 84 replites of eh HBV pnel memer tht spn the dynmi rnge of the ssy from pproximtely 1.00 log IU/mL to pproximtely 9.00 log IU/mL, for HBV s A nd C. Pnel memers 1 through 5 were HBV A, nd pnel memers 6 through 10 were HBV C. The 0.5 ml smple preprtion protool ws used for this study. This sme pnel ws lso used s prt of the site-to-site reproduiility study. One lot of mplifition regents ws run on one m2000sp nd m2000rt instrument pir y three tehniins. Eh tehniin ompleted one run per dy for seven dys, for totl of 21 runs. Four replites were run for eh pnel memer. The SD for etween-tehniin omponent nd totl SD for the Aott RelTime HBV ssy ws found to e less thn or equl to 0.06 log IU/mL nd 0.11 log IU/mL, respetively, for ll pnel memers greter thn the ssy limit of detetion (1.00 log IU/mL). The results re summrized in Tle 5. Pnel Memer n Tle 5 Aott RelTime HBV Within-Lortory Preision (Opertor-to-Opertor) Men Conentrtion (Log IU/mL) Within-Run Component SD Between- Run/Dy Component SD Between- Tehniin Component SD Totl SD,d 1 84 8.87 0.06 0.03 0.05 0.08 2 84 6.77 0.05 0.03 0.01 0.06 3 84 4.53 0.10 0.00 0.05 0.11 4 84 2.72 0.06 0.02 0.02 0.07 5 73, 0.49 0. 0.00 0.07 0.25 6 84 8.57 0.08 0.02 0.06 0.10 7 84 6.72 0.07 0.00 0.04 0.08 8 84 4.66 0.09 0.03 0.04 0.10 9 83 2.69 0.07 0.03 0.05 0.09 10 84 0.78 0.19 0.00 0.04 0.19 Stndrd Devitions (SD) re in log IU/mL. Trget not deteted for 10 smples. Error ode Internl Control Filed for one smple. d Totl preision inludes within-run, etween-run/dy, nd etween-tehniin omponents of preision. REPRODUCIBILITY IN PLASMA The plsm reproduiility pnel ws tested t three different sites y one tehnologist nd one instrument pir t eh site. Pnels tested t eh site onsisted of 40-memer pnel (10 unique pnel memers) tht inluded five onentrtion levels of one prevlent HBV genotype nd five onentrtion levels of seond prevlent HBV genotype, repeted four times within the pnel. The onentrtion levels trgeted for the reproduiility pnels spnned the liner quntittion rnge of the ssy. The HBV genotypes seleted for the reproduiility pnels were genotype A nd genotype C, reognized s prevlent in the U.S. popultion. Eh five-memer pnel ws prepred from high opy soure smple, whih ws omposed of t lest two individul ptient speimens tht hd ommon genotype. A totl of three regent lots were used. For the 0.5 ml reproduiility, eh of the three linil sites tested two of the three lots for five dys eh. Site 1 used lots A nd B, Site 2 used lots B nd C, nd Site 3 used lots A nd C. The 0.2 ml reproduiility ws tested t eh of the three linil sites using two lots for two dys eh. The SD for the etween-site omponent ws less or equl to 0.10 log IU/mL. The results re summrized in Tle 6 nd Tle 7. Tle 6 Aott RelTime HBV Reproduiility in Plsm 0.5 ml Smple Preprtion Protool Within-Run Between-Run Between-Lot Between-Site Men Con. Men Con. Component Component Component Component Totl Pnel n (Log IU/mL) (IU/mL) SD SD SD SD SD,d 1 A 0 8.93 872,502,276 0.07 0.00 0.03 0.08 0.11 2 A 119 6.84 7,087,010 0.04 0.03 0.06 0.05 0.09 3 A 0 4.70 52,574 0.09 0.02 0.06 0.10 0.15 4 A 0 2.81 665 0.05 0.02 0.06 0.08 0. 5 A 110 0.90 e 27 e 0.31 0.07 0.26 0.10 0.42 6 C 119 8.64 446,037,175 0.07 0.01 0.04 0.07 0.11 7 C 0 6.83 6,922,1 0.06 0.01 0.06 0.05 0.10 8 C 119 4.84 72,954 0.08 0.00 0.08 0.09 0.15 9 C 0 2.84 722 0.06 0.02 0.09 0.08 0.13 10 C 119 1.13 26 0.29 0.00 0.22 0.00 0.37 Invlid replite not inluded. Trget not deteted not inluded. Stndrd devitions (SD) re in log IU/mL. d The totl preision inludes within-run, etween-run, etween-lot, nd etween-site omponents of preision. e Conentrtion is elow the ssy LoD. 8
Tle 7 Aott RelTime HBV Reproduiility in Plsm 0.2 ml Smple Preprtion Protool Within-Run Between-Run Between-Lot Between-Site Men Con. Men Con. Component Component Component Component Totl Pnel n (Log IU/mL) (IU/mL) SD SD SD SD SD, 1 A 8.99 1,019,710,342 0.07 0.00 0.13 0.00 0.15 2 A 6.87 7,526,185 0.04 0.05 0.09 0.00 0. 3 A 4.70 52,678 0.06 0.08 0.14 0.00 0.17 4 A 2.83 716 0.06 0.06 0.13 0.00 0.16 5 A 37 1.03 d 21 0.40 0.00 0.18 0.02 0.44 6 C 8.64 451,101,262 0.05 0.05 0. 0.00 0.14 7 C 6.85 7,255,6 0.05 0.05 0.11 0.00 0.13 8 C 4.83 71,717 0.08 0.07 0.14 0.00 0.18 9 C 2.84 738 0.08 0.04 0.15 0.00 0.18 10 C 46 1. 26 0.30 0.00 0.27 0.00 0.41 Trget not deteted not inluded. Stndrd devitions (SD) re in log IU/mL. The totl preision inludes within-run, etween-run, etween-lot, nd etween-site omponents of preision. d Conentrtion is elow the ssy LoD. REPRODUCIBILITY IN SERUM The serum reproduiility pnel tested t eh site onsisted of 42-memer pnel (14 unique pnel memers) tht inluded seven onentrtion levels of one prevlent HBV genotype nd seven onentrtion levels of seond prevlent HBV genotype, repeted three times within the pnel. The onentrtion levels trgeted for the reproduiility pnels spnned the liner quntittion rnge of the ssy nd lso inluded some memers elow the lower limit of quntittion. The HBV genotypes seleted for the serum reproduiility pnels were genotypes tht were reognized s prevlent in the US popultion. Eh seven-memer pnel ws prepred from high opy soure smple. A totl of three regent lots were used. Eh of the three linil sites tested two of the three mplifition regent lots for five dys eh. Site 1 used lots A nd B, Site 2 used lots B nd C, nd Site 3 used lots A nd C. Eh site onduted the five dy reproduiility t oth the 0.2 ml volume nd 0.5 ml volume for two lots of mplifition regents. The results re summrized in Tle 8 nd Tle 9. Tle 8 Aott RelTime HBV Reproduiility in Serum 0.5 ml Smple Preprtion Protool Within-Run Between-Run Between-Lot Between-Site Men Con. Men Con. Component Component Component Component Totl Pnel n (Log IU/mL) (IU/mL) SD SD SD SD SD,d 1 A 89 8. 184,508,108 0.13 0.07 0.07 0.06 0.17 2 A 90 6.19 1,613,319 0.04 0.07 0.07 0.03 0.11 3 A 90 3.94 9,725 0.07 0.08 0.11 0.19 0. 4 A 89 1.96 105 0. 0.07 0.07 0.20 0.25 5 A 90 1.25 22 0.20 0.10 0. 0.20 0.32 6 A 88 1.04 13 0.17 0.10 0.08 0.18 0.28 7 A 84 0.74 e 7 e 0.22 0. 0.21 0. 0.35 8 C 90 7.22 17,211,265 0.05 0.05 0.08 0.05 0. 9 C 90 6.23 1,741,264 0.05 0.06 0.08 0.02 0. 10 C 89 3.89 9,068 0.11 0.07 0.10 0.21 0.27 11 C 89 1.64 55 0.18 0. 0.06 0.28 0.36 C 90 1.29 23 0.18 0.09 0. 0.15 0.27 13 C 88 0.95 e e 0. 0.08 0.15 0. 0.38 14 C 87 0.88 e 9 e 0.23 0.06 0.10 0.10 0.28 Invlid replite not inluded. Trget not deteted not inluded. Stndrd devitions (SD) re in log IU/mL. d The totl preision inludes within-run, etween-run, etween-lot, nd etween-site omponents of preision. e Conentrtion is elow the ssy LoD. Tle 9 Aott RelTime HBV Reproduiility in Serum 0.2 ml Smple Preprtion Protool Within-Run Between-Run Between-Lot Between-Site Men Con. Men Con. Component Component Component Component Totl Pnel n (Log IU/mL) (IU/mL) SD SD SD SD SD,d 1 A 90 8.28 205,545,691 0.16 0.04 0.03 0.00 0.17 2 A 88 6.21 1,638,140 0.04 0.04 0.03 0.00 0.06 3 A 89 3.89 8,306 0.10 0.07 0.07 0.13 0.19 4 A 89 1.90 95 0.22 0.04 0.16 0.16 0.32 5 A 88 1.10 e 16 e 0.21 0. 0.07 0.18 0.31 6 A 86, 0.89 e 10 e 0.27 0.03 0.10 0.10 0.31 7 A 82 0.62 e 7 e 0.33 0.00 0.06 0.16 0.37 8 C 90 7.22 16,6,045 0.05 0.02 0.04 0.00 0.07 9 C 90 6.25 1,776,403 0.04 0.04 0.04 0.00 0.07 10 C 90 3.84 7,550 0. 0.07 0.07 0.13 0.20 11 C 90 1.57 0.18 0.10 0.02 0.31 0.37 C 89 1.14 e 17 e 0.22 0.10 0.11 0. 0.29 13 C 87, 0.80 e 8 e 0.26 0.13 0.15 0.08 0.34 14 C 82 0.73 e 9 e 0.35 0.06 0.17 0.00 0.39 Invlid replite not inluded. Trget not deteted not inluded. Stndrd devitions (SD) re in log IU/mL. d The totl preision inludes within-run, etween-run, etween-lot, nd etween-site omponents of preision. e Conentrtion is elow the ssy LoD. 9
LIMIT OF DETECTION (LoD) Using the WHO Interntionl Stndrd The LoD of the Aott RelTime HBV ssy is 10 IU/mL for the 0.5 ml smple preprtion protool nd 15 IU/mL for the 0.2 ml smple preprtion protool. The LoD is defined s the HBV DNA onentrtion deteted with proility of 95%. The LoD ws determined y testing dilutions of the WHO Interntionl Stndrd for Heptitis B Virus DNA (NIBSC 97/746), whih were prepred in HBV negtive humn plsm nd serum. Proit nlysis of the dt ws used to determine the onentrtion of the WHO Stndrd deteted with 95% proility. The results of the LoD study in plsm nd serum t oth smple volumes re summrized in Tles 10 through 13. Tle 10 Aott RelTime HBV Limit of Detetion (LoD) in Plsm Using the WHO Interntionl Stndrd 0.5 ml Smple Preprtion Protool IU/mL Numer Tested Numer Deteted Perent Deteted 20.00 26 26 100 10.00 26 25 96 5.00 26 26 100 2.50 26 23 88 1.00 26 46 0.50 26 7 27 0.25 26 7 27 0.10 26 4 15 Proit nlysis 27 of the dt determined tht the onentrtion of HBV DNA deteted with 95% proility using the WHO Interntionl Stndrd ws 6.40 IU/mL (95% CI 3.97-13.03 IU/mL). Tle 11 Aott RelTime HBV Limit of Detetion (LoD) in Serum Using the WHO Interntionl Stndrd 0.5 ml Smple Preprtion Protool IU/mL Numer Tested Numer Deteted Perent Deteted 20.00 30 30 100 10.00 30 30 100 5.00 30 30 100 2.50 30 29 97 1.00 30 17 57 0.50 30 16 53 0.25 30 1 3 0.10 30 8 27 Proit nlysis 27 of the dt determined tht the onentrtion of HBV DNA deteted with 95% proility using the WHO Interntionl Stndrd ws 3.82 IU/mL (95% CI 1.55-69.76 IU/mL). Tle Aott RelTime HBV Limit of Detetion (LoD) in Plsm Using the WHO Interntionl Stndrd 0.2 ml Smple Preprtion Protool IU/mL Numer Tested Numer Deteted Perent Deteted 40.00 27 27 100 20.00 27 27 100 10.00 27 26 96 5.00 27 23 85 2.50 27 44 1.00 27 11 41 0.50 27 6 22 0.20 27 0 0 Proit nlysis 27 of the dt determined tht the onentrtion of HBV DNA deteted with 95% proility using the WHO Interntionl Stndrd ws 10.66 IU/mL (95% CI 7.11-19.38 IU/mL). Tle 13 Aott RelTime HBV Limit of Detetion (LoD) in Serum Using the WHO Interntionl Stndrd 0.2 ml Smple Preprtion Protool IU/mL Numer Tested Numer Deteted Perent Deteted 40.00 30 30 100 20.00 30 30 100 10.00 30 30 100 5.00 29 25 86 2.50 30 27 90 1.00 30 17 57 0.50 30 17 57 0.20 30 4 13 One replite ws exluded due to instrument error. Proit nlysis 27 of the dt determined tht the onentrtion of HBV DNA deteted with 95% proility using the WHO Interntionl Stndrd ws 5.61 IU/mL (95% CI 3.62-10.94 IU/mL). LIMIT OF DETECTION (LoD) BY GENOTYPE USING CLINICAL SPECIMENS The LoD for the ssy to detet HBV in linil speimens using the 0.5 ml smple preprtion protool volume, deteting ny of the eight genotypes tested, onsidering tht ssy does not differentite etween HBV genotypes, ws determined to e 10 IU/mL. The LoD for the ssy to detet HBV in linil speimens using the 0.2 ml smple preprtion protool volume ws determined to e 15 IU/mL. The LoD ws determined y nlysis of dilution series of ptient smples representing HBV s A, B, C, D, E, F, G, nd H nd of the WHO Interntionl Stndrd. One ptient smple for eh HBV genotype ws tested. Seril dilutions were mde in HBV serologilly negtive humn plsm nd serum to rete n eightmemer pnel with the trget onentrtions 0.10 IU/mL, 0.25 IU/mL, 0.50 IU/mL, 1.00 IU/mL, 2.50 IU/mL, 5.00 IU/mL, 10.0 IU/mL, nd 20.0 IU/mL for the 0.5 ml smple volume nd 0.20 IU/mL, 0.50 IU/mL, 1.00 IU/mL, 2.50 IU/mL, 5.00 IU/mL, 10.0 IU/mL, 20.0 IU/mL nd 40.0 IU/mL for the 0.2 ml smple volume. Proit nlysis of the dt ws used to determine the onentrtion of eh HBV genotype deteted with 95% proility. The results re summrized in Tle 14 nd Tle 15. Tle 14 Aott RelTime HBV LoD y Using Clinil Speimens 0.5 ml Smple Preprtion Protool (IU/mL) Conentrtion Deteted (95% Confidene Intervl) Tested Plsm Serum WHO 3.69 (2.59, 6.19) * A 2.31 (1.59, 4.08) 5.49 (2.86, 19.05) B 2.96 (2., 4.90) ** C 4.53 (3.18, 7.58) 3.92 (2.09, 14.50) D 3.23 (2.23, 5.62) ** E 4.73 (2.11, 39.36) 3.72 (2.55, 6.47) F 4.22 (2.80, 7.73) ** G 2.51 (1.80, 4.21) 1.94 (1.43, 3.14) H 8.11 (4.18, 27.97) ** * WHO stndrd ws not tested in serum in this study. ** s B, D, F, nd H were tested in serum with 0.2 ml volume only. See Tle 15. The LoD for the ssy to detet HBV in linil speimens using the 0.5 ml smple preprtion protool volume, deteting ny of the eight genotypes tested, onsidering tht ssy does not differentite etween HBV genotypes, ws determined to e 10 IU/mL. Tle 15 Aott RelTime HBV LoD y Using Clinil Speimens 0.2 ml Smple Preprtion Protool (IU/mL) Conentrtion Deteted (95% Confidene Intervl) Tested Plsm Serum WHO 8.16 (5.63, 13.93) * A 5.86 (4.00, 10.22) ** B 5.37 (3.72, 9.23) 2.40 (1.61, 4.65) C 8.61 (5.95, 14.68) ** D 5.34 (3.54, 9.93) 2.26 (1.55, 4.21) E 14.57 (9.63, 26.28) ** F 6.60 (4.41, 11.98) 7.18 (4.75, 13.20) G 3.84 (2.61, 6.94) ** H 10.86 (7.34, 19.10) 7.65 (5.01, 14.26) * WHO stndrd ws not tested in serum in this study. ** s A, C, E, nd G were tested in serum with 0.5 ml volume only. See Tle 14. 10
The LoD for the ssy to detet HBV in linil speimens using the 0.2 ml smple preprtion protool volume, deteting ny of the eight genotypes tested, onsidering tht ssy does not differentite etween HBV genotypes, ws determined to e 15 IU/mL. ANALYTICAL SPECIFICITY Potentilly Interfering Sustnes The suseptiility of the Aott RelTime HBV ssy to interferene y elevted levels of potentilly interfering sustnes ws evluted. HBV-negtive smples nd HBV positive smples ontining 2,933 IU/mL (3.47 log IU/mL) of HBV DNA were tested. Potentil interferene t HBV DNA onentrtions lose to the ssy LLQ (LLoQ) ws not ssessed. HBV-negtive nd positive smples were tested in plsm mtrix nd were not tested in serum. No interferene in the performne of the Aott RelTime HBV ssy ws oserved in the presene of the following endogenous sustnes for ll HBV-negtive nd positive smples tested: Hemogloin Triglyerides Biliruin Protein 500 mg/dl 3,000 mg/dl 20 mg/dl 9 g/dl Note: For hemogloin nd protein, there ws slight trend towrd lowering of the vlues of the high level HBV speimens in the presene of interfering sustnes. The men differenes of the test nd ontrol onditions for hemogloin nd protein re smll (-0.058 nd -0.1 log IU/mL, respetively) ompred to the linilly signifint differene etween two smples (1 log); s suh, these differenes re not expeted to e linilly signifint. Antivirls nd ntiiotis t onentrtions equl to or in exess of pek plsm or serum levels were tested in five pools. No interferene in the performne of the Aott RelTime HBV ssy ws oserved in the presene of the following drug pools for ll HBV-negtive nd positive smples tested: Drug Pool Drugs Tested 1 Zidovudine, Squinvir, Ritonvir, Clrithromyin, Interferon 2, Interferon 2, Didnosine 2 Avir sulfte, Amprenvir, Peginterferon 2, Peginterferon 2, Rivirin, Entevir, Adefovir 3 Tenofovir, Lmivudine, Indinvir, Gnilovir, Vlgnilovir, Aylovir, Proxetine 4 Stvudine, Efvirenz, Lopinvir, Enfuvirtide, Ciprofloxin, Fluoxetine 5 Zlitine, Nevirpine, Nelfinvir, Azithromyin, Vlylovir, Sertrline Note: A onsidertion ws mde to void omining speifi drugs within pool tht would not e used together in linil setting. Beuse the listed drugs were tested in pools, individul drug effets were not ssessed. Cross-Retivity Studies with Clinil Speimens The speifiity of the ssy ws evluted y testing 60 ptient speimens tht were positive for t lest one of the following DNA virus mrkers, RNA viruses, non-virl heptitis, or utoimmune disese sttes. Speimens tht were tested for DNA virus mrkers were in serum. Speimens tht were tested for RNA virus mrkers were in plsm or serum. HBV DNA ws not deteted in ny of the 60 speimens tested. DNA nd RNA Viruses Epstein Brr Virus (EBV) Herpes Simplex Virus 1 (HSV-1) Herpes Simplex Virus 2 (HSV-2) Cytomeglovirus (CMV) Humn Immunodefiieny Virus (HIV-1) Heptitis C Virus (HCV) Heptitis A Virus (HAV) Non-virl Heptitis nd Autoimmune Sttes Anti-nuler Antiody (ANA) Rheumtoid Ftor (RF) Cirrhosis Aloholi Heptitis Non-loholi Stetoheptitis (NASH) Autoimmune Heptitis (AUH) Heptoellulr Crinom Cross-Retivity Studies Using Nulei Aid or Virl Lyste The following viruses nd miroorgnisms were evluted for potentil ross-retivity in the Aott RelTime HBV ssy. Purified nulei id or virl lyste from eh miroorgnism or virus ws dded t onentrtion of 100,000 opies/ml to HBV DNA negtive smples nd HBV DNA positive smples tht ontined 2,933 IU/mL (3.47 log IU/mL) HBV DNA. No interferene in the performne of the Aott RelTime HBV ssy ws oserved in the presene of the potentil rossretnt miroorgnisms or viruses for ll the positive nd negtive smples tested. Miroorgnism/Virus Humn immunodefiieny virus 1 (HIV-1) Humn immunodefiieny virus 2 (HIV-2) Humn T-lymphotropi virus I (HTLV-I) Heptitis C virus (HCV) Heptitis A virus (HAV) Epstein-Brr virus (EBV) Herpes simplex virus 1 (HSV-1) Herpes simplex virus 2 (HSV-2) Cytomeglovirus (CMV) Humn herpesvirus 6B (HHV-6B) Humn herpesvirus 8 (HHV-8) Vriell-zoster virus (VZV) Vini virus (VACV) BK humn polyomvirus Humn ppillom virus 16 (HPV-16) Humn ppillom virus 18 (HPV-18) Neisseri gonorrhoee Chlmydi trhomtis Cndid lins Stphyloous ureus Stphyloous epidermidis Myoterium gordone Myoterium smegmtis Soure Virl lyste, ell ulture Virl lyste, ell ulture Virl lyste, ell ulture Virl lyste, humn speimen Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Purified nulei id Performne of the Assy with HBV-Negtive Speimens Performne of the Aott RelTime HBV ssy ws evluted y testing 1 HBV serologilly-negtive serum nd 5 HBV serologilly-negtive plsm speimens from lood donors. The speimens were tested on one m2000 instrument system with one lot of mplifition regents. HBV DNA ws not deteted for ll 9 speimens, resulting in 100% orret results: 100% (1/1) with 95% CI: 97.0% to 100% for serum smples nd 100% (5/5) with 95% CI: 97.0% to 100% for plsm smples. ANALYTICAL CARRYOVER Potentil rryover ws determined y performing three studies in whih high opy HBV positive smples were interspersed with negtive smples in hekerord pttern. For these studies, the trgeted level for the high opy HBV-positive smples ws greter thn 8 log IU/mL. The rryover rte in these representtive studies rnged from 0% to 2%. For results greter or equl to LoD, there ws n overll rryover rte of 0.63% (95% CI 0.08%-2.%). Results from the three studies re summrized in Tle 16. Study Numer of Runs Numer of Negtives Tested Tle 16 Aott RelTime HBV Anlytil Crryover Numer Deteted Perent Deteted Numer Deteted ( > LoD) Perent Deteted ( > LoD) 95% CI of Perent Deteted 1 5 100 1 1.00 0 0.00 (0.00, 3.62) 2 5 100 2 2.00 2 2.00 (0., 7.04) 3 6 0 2 1.67 0 0.00 (0.00, 3.03) Overll 16 320 5 1.56 2 0.63 (0.08, 2.) COMPARISON OF 0.2 ml VS. 0.5 ml SAMPLE PREPARATION PROTOCOLS This study used the Aott RelTime HBV ssy to quntitte HBV-positive ptient speimens. Sixty HBV-positive EDTA plsm speimens were tested in duplite with oth the 0.2 ml nd 0.5 ml smple preprtion protools. Eh duplite pir ws tested in the sme run. The dt showed slope of 0.99 nd n interept of 0.09. The study ws designed to over the dynmi rnge of the Aott RelTime HBV ssy with tul ptient smples representing genotypes (A, B, C, nd D) ommonly enountered within the US. The oserved lowest vlue in the speimen popultion for the 0.2 ml smple volume ws 1.33 log IU/mL (men vlue of the duplite pir ws 1.51 log IU/mL). For the 0.5 ml smple volume, the sme speimen hd n oserved lowest vlue of 1.40 log IU/mL (men vlue of the duplite pir ws 1.46 log IU/mL). The results re summrized in Figure 6. 11
Figure 6 Aott RelTime HBV Comprison of 0.2 ml vs. 0.5 ml Smple Preprtion Protools 0.2 ml Men Conentrtion (Log IU/mL) y = 0.99x + 0.09 r = 0.999 n = 60 0.5 ml Men Conentrtion (Log IU/mL) SERUM VS. PLASMA ACROSS THE LINEAR RANGE This study ws onduted with speimens from 30 individul HBV serologillynegtive donors. The speimens from eh donor were olleted s mthed sets in serum nd in EDTA-plsm tues. Eh pir of serum nd plsm speimens ws spiked with HBV-positive mteril t two trgeted onentrtion levels throughout the dynmi rnge of the Aott RelTime HBV Assy. Speimen types t eh trgeted onentrtion were tested one using the 0.5 ml smple preprtion protool. Two plsm-serum pirs hd quntittion vlues elow the ssy dynmi rnge nd were therefore exluded from the nlysis. Using smple size of 58, liner regression nlysis demonstrted slope of 1.00 (95% CI 0.98 to 1.01) nd n interept of 0.03 (95% CI -0.04 to 0.10). The men differene etween serum nd plsm speimens ws -0.02 log IU/mL (95% CI -0.05 to 0.01). The liner regression is shown in Figure 7. Figure 7 Aott RelTime HBV Serum vs. Plsm Aross the Liner Rnge STABILITY Speimen Stility Humn serum or plsm speimens my e stored t 15 to 30 C for up to hours or t 2 to 8 C for up to three dys. Freshly drwn whole lood (plsm or serum) speimens my e held for up to 6 hours t 2 to 30 C prior to entrifugtion. Freeze/thw effet ws tested in oth serum nd plsm for up to eight yles. Frozen speimens my e thwed t 15 to 30 C or 2 to 8 C. Thwed speimens my e stored t 2 to 8 C for up to 6 hours, if not proessed immeditely. Serum nd plsm speimens my e stored t -20 C or older for longer term storge. Stility testing results re summrized in Tle 17. Tle 17 Aott RelTime HBV Speimen Stility (Log IU/mL) Smple Type Plsm Serum Plsm (Whole Blood) Serum (Whole Blood) Plsm Freeze/Thw Serum Freeze/Thw Test Condition -26 hours t 28-32 C 72-74 hours t 2-8 C -26 hours t 28-32 C 72-74 hours t 2-8 C 6-8 hours t 28-32 C 6-8 hours t 2-8 C 6-8 hours t 28-32 C 6-8 hours t 2-8 C 8 freeze/thw yles (frozen t -20 C or older for minimum of 8 hours; thwed t 15 C to 30 C for mximum of hours) Test Condition Men Bseline Condition Men Men Differene 3.781 3.722 0.059 3.777 3.722 0.055 3.871 3.844 0.027 3.870 3.844 0.026 3.863 3.866-0.003 3.862 3.866-0.004 3.823 3.628 0.195 3.730 3.628 0.102 2.704 2.693 0.011 2.722 2.714 0.007
CLINICAL STUDIES Study Popultion The linil performne of the Aott RelTime HBV Assy for use with the m2000 System ws evluted y ssessing the ntivirl therpy response in hroni HBV infeted sujets undergoing tretment with defovir dipivoxil. The HBV DNA dt were otined from testing ptient smples previously olleted under two study protools, one of whih evluted ptients with hroni HBeAg-positive HBV infetion nd ompensted liver funtion 28 nd one tht evluted ptients with HBeAg-negtive HBV infetion with ompensted liver funtion. 29 The reltionship etween HBV DNA virl levels t vrious time points to histologi, iohemil, nd serologil responses to tretment ws determined in this study. The study popultion onsisted of hroni HBV-infeted ptients enrolled in doule lind, rndomized, pleo-ontrolled studies of defovir dipivoxil tht spnned 0 weeks. In the HBeAg-positive protool, ptients were rndomized to 10 mg defovir dipivoxil, 30 mg defovir dipivoxil, or pleo for the first weeks. Only the 10 mg defovir dipivoxil treted ptients (169 out of 171 totl) nd pleo ptients (60 rndomly seleted from 167 totl) were inluded in this study. Virl lod testing ws performed t seline nd t Weeks,, nd. The virl lod results were evluted ginst histologi, iohemil, nd serologil response t weeks. In ddition, ptients tht remined on the 10 mg defovir dipivoxil tretment were lso tested t Weeks 144, 192, 0, s ville. In the HBeAg-negtive protool, ptients were rndomized to either 10 mg defovir dipivoxil or pleo for the first weeks. The defovir dipivoxil treted ptients (3 out of 3 totl) nd pleo ptients (61 out of 61 totl) were tested t seline nd t Weeks,, nd. The virl lod results were evluted ginst histologi nd iohemil response t weeks. In ddition, ptients tht remined on the 10 mg defovir dipivoxil tretment were lso tested t Weeks 96, 144, 192, nd 0, s ville. Demogrphi dt, HBV genotype, HBeAg, nti-hbe, nd HBsAg seroonversion results, nd seline (pretretment) nd post-tretment liver iopsy results were ville. Tle 18 summrizes the sujet demogrphis. Tle 18 Sujet Demogrphis Chrteristi Ctegory Summry Sttistis HBeAg+ HBeAg- Totl Totl Numer of Sujets - N 229 184 413 Pleo - n (%) 60 (26.20) 61 (33.15) 1 10 mg defovir dipivoxil - n (%) 169 (73.80) 3 (66.85) 292 Totl Numer of Sujets with Demogrphi - n 220 184 404 Informtion Age (yr) - Medin 34 46 40 (Min, Mx) (16, 65) (18, 65) (16, 65) Weight (kg) - Medin 71 74.55 72.5 (Min, Mx) (43, 117.73) (46, 135) (43, 135) Sex Mle n (%) 164 (74.55) 152 (82.61) 316 (78.22) Femle n (%) 56 (25.45) 32 (17.39) 88 (21.78) White n (%) 80 (36.36) 2 (66.30) 202 (50.00) Re Asin n (%) 9 (58.64) 56 (30.43) 185 (45.79) Other n (%) 11 (5.00) 6 (3.26) 17 (4.21) A n (%) 64 (29.09) 11 (5.98) 75 (18.56) B n (%) 41 (18.64) 31 (16.85) 72 (17.82) C n (%) 82 (37.27) (13.04) 106 (26.) D n (%) 27 (.27) 114 (61.96) 141 (34.90) Other n (%) 6 (2.73) 4 (2.17) 10 (2.) Totl Numer of Sujets with - n 210 175 385 Knodell Sore Totl - Men (SD) 9.38 (3.29) 9.35 (3.34) 9.37 (3.31) Neroinflmmtory - Men (SD) 7.70 (2.71) 7.50 (2.75) 7.61 (2.73) Firosis - Men (SD) 1.67 (1.09) 1.86 (1.15) 1.76 (1.) Demogrphi dt were not provided for three pleo nd six tretment sujets. Tle 19 Summry of Sujets y Tretment Arm No. of Sujets - Pleo No. of Sujets - 10 mg Adefovir No. of Speimens per Sujet Totl No. of Speimens Tested Totl No. Popultion of Sujets Chroni HBeAg+ 229 60 169 2 to 7 1,036 Chroni HBeAg- 184 61 3 2 to 8 939 Totl Numer of Sujets Tested 413 1 292 2 to 8 1,975 This numer is reported s rnge euse the numer of speimens vried for eh sujet. CLINICAL STUDY RESULTS AND STATISTICAL ANALYSES Sttistil nlysis of linil dt ws used to ssess whether virl response to tretment mesured with Aott RelTime HBV Assy for use with the m2000 System is informtive for determining the response to tretment in HBeAg-positive nd HBeAg negtive ptients with hroni heptitis B. Oserving hnges in virl lod in individul ptients over time my help the liniin in the ssessment of ptient s response to therpy. Within-Sujet Vriility in Asene of Tretment The ojetive of this nlysis ws to ssess the hnge in virl lod (in log IU/mL units) etween two suessive mesurements of pleo ptients. There were 55 ptients in the pleo rm of the HBeAg-positive group nd 57 ptients in the HBeAg-negtive group tht hd ville results for oth Weeks 0 nd. These results were used to estimte within-sujet vriility, whih inludes iologil vriility s well s totl ssy vriility. The within-sujet vriility (SD) sed on these results ws estimted to e 0.79 log IU/mL for HBeAg-positive ptients nd 0.86 log IU/mL for HBeAg-negtive ptients. Biologil within-sujet vriility ws similr to the estimted within-sujet vriility sine the ssy nlytil vriility ws negligile. The medin hnge (Week Week 0) of virl lod within sujet ws estimted to e 0.00 log IU/mL for HBeAg-positive ptients nd -0.28 log IU/mL for HBeAg-negtive ptients. Approximtely 89% of the HBeAg-positive ptients nd 81% of HBeAgnegtive ptients hnge of virl lod ws less thn 2.00 log IU/mL. HBeAg-Positive Ptients Chrteriztion of Virl Lod Tle 20 nd Figure 8 illustrte the effiy, sed on HBV virl lod testing, of treting HBeAg-positive ptients with 10 mg defovir dipivoxil ompred to pleo sed on HBV virl lod testing results using Aott RelTime HBV ssy for use with the m2000 System. At Week, 22.92% (33/144) of HBeAg-positive ptients on tretment versus 0% (0/55) on pleo hd hieved very low virl lods elow 100 IU/mL. In ddition, only 23.61% (34/144) of ptients on tretment versus 81.82% (45/55) on pleo hd virl lods greter thn or equl to 10 6 IU/mL. Tle 20 Distriution of HBV Virl Lod t Week for HBeAg-Positive Ptients Adefovir Dipivoxil Pleo Virl Lod Cumultive Cumultive n % n % (IU/mL) % % TND 4 2.78 2.78 0 0.0 0.0 < 15 13 9.03 11.81 0 0.0 0.0 15 - <100 16 11.11 22.92 0 0.0 0.0 100 - < 10 3 21 14.58 37.50 2 3.64 3.64 10 3 - < 10 4 18.50 50.00 1 1.82 5.46 10 4 - < 10 5 14 9.72 59.72 4 7.27.73 10 5 - < 10 6 16.67 76.39 3 5.45 18.18 10 6 - < 10 9 32 22.22 98.61 43 78.18 96.36 10 9 2 1.39 100.00 2 3.64 100.00 Totl 144 100.00 55 100.00 TND = Trget Not Deteted The HBeAg-positive sujets were primrily Asin nd HBV s A nd C, while the HBeAg-negtive sujets were primrily White nd HBV D. Ptients inluded in the linil performne nlysis reeived either the stndrd 10 mg defovir dipivoxil dosing or pleo. Tle 19 summrizes sujets y tretment rm nd ville speimens. 13
Figure 8 demonstrtes the medin virl lod hnge nd inter-qurtile rnge of hnge from seline for HBeAg-positve sujets on tretment ompred to pleo. This shows the impt of defovir dipivoxil tretment on the virl lod of HBeAg-positive ptients with hroni heptitis B. Figure 8 Medin nd Inter-Qurtile Rnge of Chnge in HBV DNA from Bseline: HBeAg-Positive Sujets The effet of therpy for ptients with hroni HBV infetion n e ssessed y mesuring the HBV DNA (expeted redution to low or undetetle levels), nd monitoring for virl reound tht ould e ssoited with resistne. Results in Tle 21 show tht 70.41% (119/169) of the treted sujets hieved ndir, or lowest onentrtion, virl lod level y Week. Of the 49 sujets tht hieved ndir y Week, 13.79% (4/29) of the sujets hd greter thn or equl to one log IU/mL inrese y Week (20 of these sujets did not hve Week result). Tle 21 Distriution of HBeAg-Positive Sujets y Week on Tretment nd the Virl Lod t Whih the Ndir ws Rehed Numer (%) of Ptients With the Ndir Virl Lod Ahieved y Week Ndir Virl Lod Totl By Cumultive (IU/mL) 144 192 0 Virl Lod By Virl Lod TND 0 (0.00) 0 (0.00) 4 (2.37) 1 (0.59) 1 (0.59) 4 (2.37) 10 (5.92) 10 (5.92) < 15 0 (0.00) 1 (0.59) 13 (7.69) 1 (0.59) 6 (3.55) 1 (0.59) 22 (13.02) 32 (18.94) 15 - < 100 0 (0.00) 0 (0.00) (7.10) 3 (1.78) 4 (2.37) 2 (1.18) 21 (.43) 53 (31.37) 100 - < 10 3 1 (0.59) 3 (1.78) 15 (8.88) 0 (0.00) 1 (0.59) 7 (4.14) 27 (15.98) 80 (47.35) 10 3 - < 10 4 3 (1.78) 8 (4.73) 5 (2.96) 1 (0.59) 1 (0.59) 2 (1.18) 20 (11.83) 100 (59.18) 10 4 - < 10 5 3 (1.78) 5 (2.96) 7 (4.14) 3 (1.78) 1 (0.59) 2 (1.18) 21 (.43) 1 (71.61) 10 5 - < 10 6 3 (1.78) 5 (2.96) 8 (4.73) 1 (0.59) 3 (1.78) 2 (1.18) 22 (13.02) 143 (84.63) 10 6 - < 10 9 8 (4.73) 9 (5.33) 6 (3.55) 1 (0.59) 0 (0.00) 2 (1.18) 26 (15.38) 169 (100.00) Totl By Week 18 (10.65) 31 (18.34) 70 (41.42) 11 (6.51) 17 (10.06) 22 (13.02) Cumultive By Week 18 (10.65) 49 (28.99) 119 (70.41) 130 (76.92) 147 (86.98) 169 (100.00) TND = Trget Not Deteted Two ptients out of 169 hieved HBsAg seroonversion. One ptient hd results showing HBsAg seronversion t oth Weeks 192 nd 0. The other ptient hieved seroonversion t Week 0. These two ptients were white mles, HBV genotype A, nd > 30 yers of ge. A summry of these results is provided in Tle 22. Tle 22 HBeAg-Positive Sujets with HBsAg Seroonversion Conentrtion (Log IU/mL) Week 0 Week Week Week Week 144 Week 192 Week 0 Sujet 1 6.99 4.98 2.08 1.50 TND TND TND Sujet 2 8.59 6.57 6.85 6.72 5.68 1.45 * TND = Trget Not Deteted * The Aott RelTime HBV result for the Week 0 time point ws exluded due to tehniin error. 14
Summries of the effet of seline ovrites for the HBeAg-positive popultion re provided in Tle 23 through Tle 25. Tle 23 Assoition Between s to Tretment t Week nd Bseline Covrites for HBeAg-Positive Ptients to Tretment Covrite Ctegory N No. of Ptients with Proportion (%) of Ptients with Re Asin Other 75 52 47 28 62.67 53.85 l > 30 52 75 34 41 65.38 54.67 Sex Mle Femle 97 30 58 17 59.79 56.67 Age Non- 72 55 47 28 65.28 50.91 Re Asin Other 82 51 47 22 57.32 43.14 Biohemil > 30 56 77 33 36 58.93 46.75 Sex Mle Femle 102 31 53 16 51.96 51.61 Age Non- 79 54 45 56.96 44.44 Re Asin Other 84 55 21 15 25.00 27.27 HBeAg Loss > 30 59 80 13 23 22.03 28.75 Sex Mle Femle 105 34 26 10.76 29.41 Age Non- 80 59 19 17 23.75 28.81 Re Asin 84 8 9.52 Other 55 10 18.18 Sex HBeAg Seroonversion Mle Femle 105 34 14 4 13.33 11.76 Age > 30 59 80 7 11 11.86 13.75 80 6 7.50 Non- 59 20.34 Undjusted Odds Rtio 1.44 (0.66, 3.14) 1.14 (0.45, 2.81) 1.57 (0.71, 3.49) 1.81 (0.83, 3.95) 1.77 (0.82, 3.82) 1.01 (0.42, 2.45) 1.63 (0.77, 3.) 1.65 (0.78, 3.53) 0.89 (0.38, 2.09) 0.79 (0.31, 2.11) 0.70 (0.29, 1.63) 0.77 (0.34, 1.78) 0.47 (0.15, 1.45) 1.15 (0.33, 5.18) 0.84 (0.26, 2.58) 0.32 (0.09, 1.00) The sttistil signifine of the ssoitions of the Re, Sex, Age nd ovrites with virl response ws studied, nd the results re summrized in Tles nd Tle 25. All lower limits of the 95% onfidene intervls in Tle re smller thn 1, exept for Re nd t Weeks nd (when virl response is defined s < 2,000 IU/mL). When response is defined s < 2,000 IU/mL, logisti regression nlyses resulted in no sttistilly signifint ssoitions etween the four ovrites nd virl lod. All lower limits of the 95% onfidene intervls in Tle 25 re smller thn 1 (when virl response is defined s 2 log derese). When response is defined s 2 log derese, logisti regression nlyses resulted in only gender t Week showing orderline sttistilly signifint ssoition (p = 0.043) with virl lod. Generlly, the virologil responses t Weeks, nd do not pper to e orrelted with Re, Sex, Age, nd HBV. Tle Odds Rtios for the Assoition Between Virl (< 2,000 IU/mL) nd Covrites, y Week, for n HBeAg-Positive Popultion Covrite Ctegory Week N No. Below 2,000 IU/mL Proportion (%) Below 2,000 IU/mL Asin 87 21.14 Other 61 6 9.84 Re Asin 81 30 37.04 Other 58 9 15.52 Asin 79 34 43.04 Other 52 17 32.69 Mle 1 20 17.86 Femle 36 7 19.44 Sex Mle 104 27 25.96 Femle 35 34.29 Mle 100 37 37.00 Femle 31 14 45.16 63 10 15.87 > 30 85 17 20.00 Age 61 18 29.51 > 30 78 21 26.92 55 21 38.18 > 30 76 30 39.47 81 20.69 Non- 67 7 10.45 76 29 38.16 Non- 63 10 15.87 74 32 43. Non- 57 19 33.33 Undjusted Odds Rtio 2.92 (1.04, 9.41) 3.20 (1.30, 8.42) 1.56 (0.71, 3.47) 0.90 (0.32, 2.78) 0.67 (0.28, 1.70) 0.71 (0.29, 1.76) 0.75 (0.28, 1.92) 1.14 (0.50, 2.55) 0.95 (0.44, 2.05) 2.81 (1.04, 8.41) 3.27 (1.36, 8.29) 1.52 (0.70, 3.34) 15
Tle 25 Odds Rtios for the Assoition Between Virl ( 2 Log Derese From Bseline Result) nd Covrites, y Week, for n HBeAg-Positive Popultion Covrite Ctegory Week N No. with 2 Log Derese Proportion (%) with 2 Log Derese Asin 87 54 62.07 Other 61 40 65.57 Re Asin 81 62 76.54 Other 58 42 72.41 Asin 79 56 70.89 Other 52 39 75.00 Mle 1 66 58.93 Femle 36 28 77.78 Sex Mle 104 74 71.15 Femle 35 30 85.71 Mle 100 71 71.00 Femle 31 77.42 63 41 65.08 > 30 85 53 62.35 Age 61 44 72.13 > 30 78 60 76.92 55 40 72.73 > 30 76 55 72.37 81 51 62.96 Non- 67 43 64.18 76 58 76.32 Non- 63 46 73.02 74 52 70.27 Non- 57 43 75.44 Undjusted Odds Rtio 0.86 (0.41, 1.79) 1. (0.53, 2.88) 0.81 (0.33, 1.91) 0.41 (0.15, 1.03) 0.41 (0.11, 1.22) 0.71 (0.23, 1.96) 1.13 (0.54, 2.36) 0.78 (0.34, 1.81) 1.02 (0.44, 2.41) 0.95 (0.46, 1.96) 1.19 (0.51, 2.75) 0.77 (0.32, 1.80) Positive Preditive Vlue (PPV), Negtive Preditive Vlue (NPV), nd Odds Rtio (OR) Anlysis in HBeAg-Positive Popultion For eh ptient, the linil responses:, Biohemil, HBeAg Loss, Anti-HBe Gin, nd Seroonversion were mesured t vrious times on tretment. These linil responses were defined s follows: response - improvement of histologi sttus y t lest 2 units of the Knodell nero-inflmmtory sore without deteriortion of the firosis sore ompred to the histologi sttus t seline Biohemil response - normliztion of ALT test result ompred to the iohemil sttus t seline HBeAg Loss - HBeAg undetetle Anti-HBe Gin - ntiody ginst HBeAg deteted Seroonversion - HBeAg undetetle nd ntiody ginst HBeAg deteted Additionlly, HBsAg seroonversion dt were olleted. Two ptients out of 169 hieved HBsAg seroonversion. One ptient hd results showing HBsAg seronversion t oth Weeks 192 nd 0. The other ptient hieved seroonversion t Week 0. These two ptients were white mles, HBV genotype A, nd > 30 yers of ge. A summry of these results is provided in Tle 22. Virl lod response ws defined s either HBV DNA less thn 2,000 IU/mL or greter thn or equl to 2 log IU/mL derese from seline. Sttistil nlysis (PPV) ws performed to evlute the ssoition etween the linil responses t Weeks, 144, 192, or 0 nd virl lod response t Weeks,, or of tretment. Sttistil nlysis (NPV) ws performed to evlute whether there is n ssoition etween the linil non-responses t Weeks, 144, 192, or 0 nd virl lod non-response t Weeks,, or of tretment. Virl < 2,000 IU/mL As shown in Tle 26, erly virl response (Weeks,, ) is informtive in prediting linil responses t Week. The PPV is the highest for the ssoition of virl response nd the histologi nd iohemil responses; while NPV is the highest for the ssoition of virl response nd the serologil responses (HBeAg loss, nti-hbe gin, nd seroonversion). Virl response t Weeks,, nd is informtive in prediting iohemil, HBeAg loss, nti-hbe gin, nd seroonversion t Week (i.e., the lower 95% CI limits for the odds rtio exeed 1.0). Virl response t Week is lso informtive in prediting histologi improvement t Week. Virl response t Week is informtive in prediting HBeAg loss t Week 144 nd virl response t Week is lso informtive in prediting nti-hbe gin nd seroonversion t Week 0 of tretment. 16
Week of Virl Week of Clinil 144 144 144 192 192 192 0 0 0 Tle 26 PPV, NPV, nd Odds Rtio for Individul Clinil s During Tretment Predited y Erly Virl (< 2,000 IU/mL) in HBeAg-Positive Sujets Clinil PPV (%) (Proportion) PPV NPV Odds Rtio NPV (%) (Proportion) 79.2 (19/) (57.3, 92.1) 43.3 (39/90) (33.1, 54.2) 2.91 (0.93, 10.76) Biohemil 84.0 (21/25) (63.1, 94.7) 55.3 (52/94) (44.7, 65.5) 6.50 (1.95, 27.66) HBeAg Loss 64.0 (16/25) (42.6, 81.3) 82.8 (82/99) (73.6, 89.4) 8.58 (2.94, 25.54) Anti-HBe Gin 36.0 (9/25) (18.7, 57.4) 91.9 (91/99) (84.2, 96.2) 6.40 (1.85, 21.90) Seroonversion 36.0 (9/25) (18.7, 57.4) 91.9 (91/99) (84.2, 96.2) 6.40 (1.85, 21.90) 80.0 (28/35) (62.5, 90.9) 49.3 (37/75) (37.7, 61.0) 3.89 (1.42, 11.76) Biohemil 83.8 (31/37) (67.3, 93.2) 59.7 (46/77) (47.9, 70.6) 7.67 (2.68,.74) HBeAg Loss 62.2 (23/37) (44.8, 77.1) 90.2 (74/82) (81.2, 95.4) 15.20 (5.15, 46.52) Anti-HBe Gin.3 (9/37) (.4, 41.6) 92.7 (76/82) (84.2, 97.0) 4.07 (1.16, 15.06) Seroonversion.3 (9/37) (.4, 41.6) 92.7 (76/82) (84.2, 97.0) 4.07 (1.16, 15.06) 74.0 (37/50) (59.4, 84.9) 49.3 (35/71) (37.3, 61.3) 2.77 (1.19, 6.62) Biohemil 78.0 (39/50) (63.7, 88.0) 63.2 (/76) (51.3, 73.7) 6.08 (2.52, 15.15) HBeAg Loss 62.7 (32/51) (.1, 75.5) 97.5 (78/80) (90.4, 99.6) 65.68 (14.10, 587.82) Anti-HBe Gin 33.3 (17/51) (21.1,.0) 100.0 (80/80) (94.3, 100.0) >39.50 d Seroonversion 33.3 (17/51) (21.1,.0) 100.0 (80/80) (94.3, 100.0) >39.50 d not ville Biohemil 66.7 (4/6) (.1, 94.0) 55.6 (25/45) (40.1, 70.0) 2.50 (0.31, 29.77) HBeAg Loss 50.0 (3/6) (13.9, 86.1) 84.4 (38/45) (69.9, 93.0) 5.43 (0.58, 47.41) Anti-HBe Gin 33.3 (2/6) (6.0, 75.9) 86.7 (39/45) (72.5, 94.5) 3.25 (0., 28.53) Seroonversion 33.3 (2/6) (6.0, 75.9) 86.7 (39/45) (72.5, 94.5) 3.25 (0., 28.53) not ville Biohemil 70.0 (7/10) (35.4, 91.9) 57.5 (23/40) (41.0, 72.6) 3.16 (0.60, 21.20) HBeAg Loss 50.0 (5/10) (20.1, 79.9) 87.5 (35/40) (72.4, 95.3) 7.00 (1.11, 42.84) Anti-HBe Gin 30.0 (3/10) (8.1, 64.6) 87.5 (35/40) (72.4, 95.3) 3.00 (0.37, 19.68) Seroonversion 30.0 (3/10) (8.1, 64.6) 87.5 (35/40) (72.4, 95.3) 3.00 (0.37, 19.68) not ville Biohemil 63.6 (7/11) (31.6, 87.6) 58.3 (21/36) (40.9, 74.0) 2.45 (0.50, 13.33) HBeAg Loss 36.4 (4/11) (.4, 68.4) 86.1 (31/36) (69.7, 94.8) 3.54 (0.54, 21.21) Anti-HBe Gin 27.3 (3/11) (7.3, 60.7) 88.9 (32/36) (73.0, 96.4) 3.00 (0.36, 21.45) Seroonversion 27.3 (3/11) (7.3, 60.7) 88.9 (32/36) (73.0, 96.4) 3.00 (0.36, 21.45) not ville Biohemil 60.0 (3/5) (17.0, 92.7) 39.5 (15/38) (.5, 56.6) 0.98 (0.10, 13.02) HBeAg Loss 40.0 (2/5) (7.3, 83.0) 57.9 (22/38) (40.9, 73.3) 0.92 (0.07, 9.02) Anti-HBe Gin 40.0 (2/5) (7.3, 83.0) 76.3 (29/38) (59.4, 88.0) 2.15 (0.15, 21.62) Seroonversion 40.0 (2/5) (7.3, 83.0) 76.3 (29/38) (59.4, 88.0) 2.15 (0.15, 21.62) not ville Biohemil 66.7 (6/9) (30.9, 91.0) 40.6 (13/32) (.2, 59.2) 1.37 (0., 9.92) HBeAg Loss 44.4 (4/9) (15.3, 77.3) 62.5 (20/32) (43.7, 78.3) 1.33 (0.22, 7.58) Anti-HBe Gin 33.3 (3/9) (9.0, 69.1) 78.1 (25/32) (59.6, 90.1) 1.79 (0.23, 11.22) Seroonversion 33.3 (3/9) (9.0, 69.1) 78.1 (25/32) (59.6, 90.1) 1.79 (0.23, 11.22) not ville Biohemil 77.8 (7/9) (40.2, 96.1) 37.5 (/32) (21.7, 56.3) 2.10 (0.32, 23.57) HBeAg Loss 66.7 (6/9) (30.9, 91.0) 62.5 (20/32) (43.7, 78.3) 3.33 (0.56, 23.78) Anti-HBe Gin 55.6 (5/9) (22.7, 84.7) 81.3 (26/32) (63.0, 92.1) 5.42 (0.83, 35.32) Seroonversion 55.6 (5/9) (22.7, 84.7) 81.3 (26/32) (63.0, 92.1) 5.42 (0.83, 35.32) * (0/0) * 33.3 (3/9) (9.0, 69.1) * Biohemil 66.7 (2/3) (.5, 98.2) 29.0 (9/31) (14.9,.2) 0.82 (0.04, 53.57) HBeAg Loss 66.7 (2/3) (.5, 98.2) 58.1 (18/31) (39.3, 74.9) 2.77 (0.13, 172.28) Anti-HBe Gin 33.3 (1/3) (1.8, 87.5) 86.7 (26/30) (68.4, 95.6) 3.25 (0.04, 73.98) Seroonversion 33.3 (1/3) (1.8, 87.5) 87.1 (27/31) (69.2, 95.8) 3.38 (0.05, 76.68) 0.0 (0/1) (0.0, 94.5) 37.5 (3/8) (10.2, 74.1) 0.00 (0.00, 15.20) Biohemil 60.0 (3/5) (17.0, 92.7) 29.6 (8/27) (14.5, 50.3) 0.63 (0.06, 9.06) HBeAg Loss 60.0 (3/5) (17.0, 92.7) 63.0 (17/27) (42.5, 79.9) 2.55(0., 34.44) Anti-HBe Gin 20.0 (1/5) (1.1, 70.1) 84.6 (22/26) (64.3, 95.0) 1.38 (0.02, 20.07) Seroonversion 20.0 (1/5) (1.1, 70.1) 85.2 (23/27) (65.4, 95.1) 1.44 (0.02, 20.91) 100.0 (1/1) (5.5, 100.0) 57.1 (4/7) (20.2, 88.2) * Biohemil 83.3 (5/6) (36.5, 99.1) 30.8 (8/26) (15.1, 51.9) 2.22 (0.19, 118.05) HBeAg Loss 83.3 (5/6) (36.5, 99.1) 65.4 (17/26) (44.4, 82.1) 9.44 (0.81, 472.23) Anti-HBe Gin 50.0 (3/6) (13.9, 86.1) 96.0 (/25) (77.7, 99.8).00 (1.21, 1,309.0) Seroonversion 50.0 (3/6) (13.9, 86.1) 96.2 (25/26) (78.4, 99.8) 25.00 (1.26, 1,361.3) * Undefined (division y zero) Shding indites sttistil signifine. Seroonversion - HBeAg undetetle nd ntiody ginst HBeAg deteted An ssoition with, rther thn predition of, linil responses is demonstrted when mesuring the virl response t Week. d The odds rtio lultions re undefined when NPV is 100% or PPV is 100% or missing. Where the denomintors for oth the NPV nd PPV re greter thn five, minimum odds rtio ws determined y sutrting one speimen from the numertor of the 100% prmeter estimte (NPV or PPV). 17
Tles 27 nd 28 demonstrte tht the NPV is very high (greter thn 89% for Week of linil response) for the ssoition of erly virl response with the omintion of ll three responses (histologi, iohemil, nd serologil - HBeAg loss or seroonversion). These dt indite tht HBeAg-positive sujets without n erly virl response (defined s < 2,000 IU/mL derese) re very unlikely to hieve ll three linil responses y Week, s result of tretment. The numer of sujets t Week 0 ws smll, nd therefore ville dt is indequte to drw onlusions out the ssoition of the erly virl response with linil responses t lter weeks. Tle 27 PPV, NPV, nd Odds Rtio (OR) for Comintion of, Biohemil, nd HBeAg Loss s During Tretment Predited y n Erly Virl (< 2,000 IU/mL) in HBeAg-Positive Sujets Week of Virl Week of Clinil PPV (%) (Proportion) PPV NPV (%) (Proportion) NPV Odds Rtio 41.7 (10/) (22.8, 63.1) 89.9 (80/89) (81.2, 95.0) 6.35 (1.90, 20.95) 42.9 (15/35) (26.8, 60.5) 97.3 (72/74) (89.7, 99.5) 27.00 (5.38, 252.97) 36.7 (18/49) (23.8, 51.7) 98.6 (70/71) (91.3, 99.9) 40.65 (5.75, 1716.7) 0 * (0/0) * 55.6 (5/9) (22.7, 84.7) * 0 0.0 (0/1) (0.0, 94.5) 62.5 (5/8) (25.9, 89.8) 0.00 (0.00, 38.00) 0 100.0 (1/1) (5.5, 100.0) 71.4 (5/7) (30.3, 94.9) * * Undefined (division y zero) Shding indites sttistil signifine. An ssoition with, rther thn predition of, linil responses is demonstrted when mesuring the virl response t Week. Tle 28 PPV, NPV, nd Odds Rtio (OR) for Comintion of, Biohemil, nd Seroonversion s During Tretment Predited y n Erly Virl (< 2,000 IU/mL) in HBeAg-Positive Sujets Week of Virl Week of Clinil PPV (%) (Proportion) PPV NPV (%) (Proportion) NPV Odds Rtio 25.0 (6/) (10.6, 47.1) 97.8 (87/89) (91.4, 99.6) 14.50 (2.28, 152.63) 17.1 (6/35) (7.2, 34.3) 98.6 (73/74) (91.7, 99.9) 15.10 (1.68, 703.89) 16.3 (8/49) (7.8, 30.2) 100.0 (71/71) (93.6, 100.0) >13.66 0 * (0/0) * 77.8 (7/9) (40.2, 96.1) * 0 0.0 (0/1) (0.0, 94.5) 75.0 (6/8) (35.6, 95.5) 0.00 (0.00, 66.50) 0 100.0 (1/1) (5.5, 100.0) 100.0 (7/7) (56.1, 100.0) * * Undefined (division y zero) Shding indites sttistil signifine. An ssoition with, rther thn predition of, linil responses is demonstrted when mesuring the virl response t Week. The odds rtio lultions re undefined when NPV is 100% or PPV is 100% or missing. Where the denomintors for oth the NPV nd PPV re greter thn five, minimum odds rtio ws determined y sutrting one speimen from the numertor of the 100% prmeter estimte (NPV or PPV). 18
Virl 2 Log IU/mL Derese As shown in Tle 29, erly virl response (Weeks,, ) is informtive in prediting linil responses t Week. High NPV (>90.9%) is oserved for the ssoition of virl response nd the serologil responses (HBeAg loss, nti-hbe Gin, nd seroonversion). The signifine of virl response t Weeks,, nd in prediting histologi, iohemil, HBeAg loss, nti-hbe gin, nd seroonversion t Week nd lter time points is ssessed y the lower 95% CI limit for the odds rtio exeeding 1.0. Week of Virl Week of Clinil Tle 29 PPV, NPV, nd Odds Rtio for Individul Clinil s During Tretment Predited y Erly Virl ( 2 Log IU/mL Derese) in HBeAg-Positive Sujets Clinil PPV (%) (Proportion) PPV NPV (%) (Proportion) NPV Odds Rtio 67.5 (52/77) (55.8, 77.5) 51.4 (19/37) (34.7, 67.8) 2.20 (0.91, 5.28) Biohemil 58.2 (46/79) (46.6, 69.1) 57.5 (23/40) (41.0, 72.6) 1.89 (0.82, 4.39) HBeAg Loss 36.3 (29/80) (26.0, 47.8) 90.9 (40/44) (77.4, 97.0) 5.69 (1.76, 23.79) Anti-HBe Gin 18.8 (15/80) (11.2, 29.4) 95.5 (42/44) (83.3, 99.2) 4.85 (1.03, 45.38) Seroonversion 18.8 (15/80) (11.2, 29.4) 95.5 (42/44) (83.3, 99.2) 4.85 (1.03, 45.38) 62.7 (52/83) (51.3, 72.8).1 (13/27) (29.2, 67.6) 1.56 (0.59, 4.09) Biohemil 59.3 (51/86) (.2, 69.6) 60.7 (17/28) (40.7, 77.9) 2.25 (0.87, 5.98) HBeAg Loss 33.3 (30/90) (.0, 44.1) 96.6 (28/29) (80.4, 99.8) 14.00 (2.07, 591.20) Anti-HBe Gin 16.7 (15/90) (9.9, 26.3) 100.0 (29/29) (85.4, 100.0) >5.60 d Seroonversion 16.7 (15/90) (9.9, 26.3) 100.0 (29/29) (85.4, 100.0) >5.60 d 66.7 (60/90) (55.9, 76.0) 58.1 (18/31) (39.3, 74.9) 2.77 (1.11, 7.00) Biohemil 63.4 (59/93) (52.8, 73.0) 75.8 (25/33) (57.4, 88.3) 5.42 (2.06, 15.32) HBeAg Loss 35.8 (34/95) (26.4, 46.3) 100.0 (36/36) (88.0,100.0) >19.51 d Anti-HBe Gin 17.9 (17/95) (11.1, 27.4) 100.0 (36/36) (88.0,100.0) >7.63 d Seroonversion 17.9 (17/95) (11.1, 27.4) 100.0 (36/36) (88.0,100.0) >7.63 d not ville Biohemil 43.3 (13/30) (26.0, 62.3) 47.6 (10/21) (26.4, 69.7) 0.70 (0.20, 2.46) 144 HBeAg Loss 23.3 (7/30) (10.6, 42.7) 85.7 (18/21) (62.6, 96.2) 1.83 (0.35,.35) Anti-HBe Gin 16.7 (5/30) (6.3, 35.5) 85.7 (18/21) (62.6, 96.2) 1.20 (0.20, 8.70) Seroonversion 16.7 (5/30) (6.3, 35.5) 85.7 (18/21) (62.6, 96.2) 1.20 (0.20, 8.70) not ville Biohemil 44.7 (17/38) (29.0, 61.5) 41.7 (5/) (16.5, 71.4) 0.58 (0., 2.59) 144 HBeAg Loss 26.3 (10/38) (14.0, 43.4) 100.0 (/) (69.9, 100.0) >3.93 d Anti-HBe Gin 21.1 (8/38) (10.1, 37.8) 100.0 (/) (69.9, 100.0) >2.93 d Seroonversion 21.1 (8/38) (10.1, 37.8) 100.0 (/) (69.9, 100.0) >2.93 d not ville Biohemil.3 (14/29) (29.9, 67.1) 55.6 (10/18) (31.3, 77.6) 1.17 (0.31, 4.49) 144 HBeAg Loss 20.7 (6/29) (8.7, 40.3) 83.3 (15/18) (57.7, 95.6) 1.30 (0.23, 9.25) Anti-HBe Gin 17.2 (5/29) (6.5, 36.5) 88.9 (16/18) (63.9, 98.1) 1.67 (0.23, 19.34) Seroonversion 17.2 (5/29) (6.5, 36.5) 88.9 (16/18) (63.9, 98.1) 1.67 (0.23, 19.34) not ville Biohemil 60.0 (15/25) (38.9, 78.2) 38.9 (7/18) (18.3, 63.9) 0.95 (0.23, 3.89) 192 HBeAg Loss.0 (/25) (28.3, 68.2) 66.7 (/18) (41.2, 85.6) 1.85 (0.45, 7.96) Anti-HBe Gin 28.0 (7/25) (.9, 49.6) 77.8 (14/18) (51.9, 92.6) 1.36 (0.27, 7.62) Seroonversion 28.0 (7/25) (.9, 49.6) 77.8 (14/18) (51.9, 92.6) 1.36 (0.27, 7.62) not ville Biohemil 56.7 (17/30) (37.7, 74.0) 27.3 (3/11) (7.3, 60.7) 0.49 (0.07, 2.64) 192 HBeAg Loss 43.3 (13/30) (26.0, 62.3) 72.7 (8/11) (39.3, 92.7) 2.04 (0.38, 14.07) Anti-HBe Gin 33.3 (10/30) (17.9, 52.9) 100.0 (11/11) (67.9, 100.0) >5.00 d Seroonversion 33.3 (10/30) (17.9, 52.9) 100.0 (11/11) (67.9, 100.0) >5.00 d not ville Biohemil 76.9 (20/26) (55.9, 90.2) 53.3 (8/15) (27.4, 77.7) 3.81 (0.80, 18.49) 192 HBeAg Loss 46.2 (/26) (27.1, 66.3) 60.0 (9/15) (32.9, 82.5) 1.29 (0.30, 5.77) Anti-HBe Gin 34.6 (9/26) (17.9, 55.6) 86.7 (13/15) (58.4, 97.7) 3.44 (0.55, 37.07) Seroonversion 34.6 (9/26) (17.9, 55.6) 86.7 (13/15) (58.4, 97.7) 3.44 (0.55, 37.07) 100.0 (5/5) (46.3, 100.0) 75.0 (3/4) (21.9, 98.7) * Biohemil 73.7 (14/19) (.6, 89.9) 33.3 (5/15) (13.0, 61.3) 1.40 (0.25, 7.92) 0 HBeAg Loss 57.9 (11/19) (34.0, 78.9) 73.3 (11/15) (44.8, 91.1) 3.78 (0.72, 21.84) Anti-HBe Gin 27.8 (5/18) (10.7, 53.6) 100.0 (15/15) (74.7, 100.0) >5.38 d Seroonversion 26.3 (5/19) (10.1, 51.4) 100.0 (15/15) (74.7, 100.0) >5.00 d 71.4 (5/7) (30.3, 94.9) 100.0 (2/2) (19.8, 100.0) * Biohemil 69.6 (16/23) (47.0, 85.9) 33.3 (3/9) (9.0, 69.1) 1.14 (0.14, 7.) 0 HBeAg Loss 52.2 (/23) (31.1, 72.6) 88.9 (8/9) (50.7, 99.4) 8.73 (0.86, 418.80) Anti-HBe Gin 22.7 (5/22) (8.7, 45.8) 100.0 (9/9) (62.9, 100.0) >2.35 d Seroonversion 21.7 (5/23) (8.3, 44.2) 100.0 (9/9) (62.9, 100.0) >2.22 d 66.7 (4/6) (.1, 94.0) 100.0 (2/2) (19.8, 100.0) * Biohemil 77.3 (17/22) (54.2, 91.3) 40.0 (4/10) (13.7, 72.6) 2.27 (0.32, 14.76) 0 HBeAg Loss 54.5 (/22) (32.7, 74.9) 80.0 (8/10) (44.2, 96.5) 4.80 (0.69, 53.92) Anti-HBe Gin 19.0 (4/21) (6.3, 42.6) 100.0 (10/10) (65.5, 100.0) >2. d Seroonversion 18.2 (4/22) (6.0, 41.0) 100.0 (10/10) (65.5, 100.0) >2.00 d * Undefined (division y zero) Shding indites sttistil signifine. Seroonversion - HBeAg undetetle nd ntiody ginst HBeAg deteted. An ssoition with, rther thn predition of, linil responses is demonstrted when mesuring the virl response t Week. d The odds rtio lultions re undefined when NPV is 100% or PPV is 100% or missing. Where the denomintors for oth the NPV nd PPV re greter thn five, minimum odds rtio ws determined y sutrting one speimen from the numertor of the 100% prmeter estimte (NPV or PPV). 19
Tles 30 nd 31 demonstrte tht the NPV is high (greter thn or equl to 97% for response t Week ) for the ssoition of erly virl response with the omintion of ll three responses (histologi, iohemil, nd serologil - HBeAg loss or seroonversion). These dt indite tht HBeAg-positive sujets without n erly virl response (defined s 2 log IU/mL derese) re unlikely to hieve ll three linil responses y Week, s result of tretment. The numer of sujets t Week 0 ws smll, nd therefore ville dt is indequte to drw onlusions out the ssoition of the erly virl response with linil responses t this time point. Tle 30 PPV, NPV, nd Odds Rtio (OR) for Comintion of, Biohemil, nd HBeAg Loss s During Tretment Predited y n Erly Virl ( 2 Log IU/mL Derese) in HBeAg-Positive Sujets Week of Virl Week of Clinil PPV NPV PPV (%) (Proportion) NPV (%) (Proportion) Odds Rtio 23.7 (18/76) (15.0, 35.1) 97.3 (36/37) (84.2, 99.9) 11.17 (1.60, 478.17) 20.7 (17/82) (.9, 31.4) 100.0 (27/27) (84.5, 100.0) >6.80 21.3 (19/89) (13.7, 31.6) 100.0 (31/31) (86.3, 100.0) >8.14 0 60.0 (3/5) (17.0, 92.7) 75.0 (3/4) (21.9, 98.7) 4.50 (0.15, 313.49) 0 42.9 (3/7) (11.8, 79.8) 100.0 (2/2) (19.8, 100.0) * 0 50.0 (3/6) (13.9, 86.1) 100.0 (2/2) (19.8, 100.0) * * Undefined (division y zero) Shding indites sttistil signifine. An ssoition with, rther thn predition of, linil responses is demonstrted when mesuring the virl response t Week. The odds rtio lultions re undefined when NPV is 100% or PPV is 100% or missing. Where the denomintors for oth the NPV nd PPV re greter thn five, minimum odds rtio ws determined y sutrting one speimen from the numertor of the 100% prmeter estimte (NPV or PPV). Tle 31 PPV, NPV, nd Odds Rtio (OR) for Comintion of, Biohemil, nd Seroonversion s During Tretment Predited y n Erly Virl ( 2 Log IU/mL Derese) in HBeAg-Positive Sujets Week of Virl Week of Clinil PPV NPV PPV (%) (Proportion) NPV (%) (Proportion) Odds Rtio 10.5 (8/76) (5.0, 20.2) 100.0 (37/37) (88.3, 100.0) >4. 8.5 (7/82) (3.8, 17.3) 100.0 (27/27) (84.5, 100.0) >2.43 9.0 (8/89) (4.2, 17.4) 100.0 (31/31) (86.3, 100.0) >2.96 0 40.0 (2/5) (7.3, 83.0) 100.0 (4/4) (39.6, 100.0) * 0 28.6 (2/7) (5.1, 69.7) 100.0 (2/2) (19.8, 100.0) * 0 16.7 (1/6) (0.9, 63.5) 100.0 (2/2) (19.8, 100.0) * * Undefined (division y zero) An ssoition with, rther thn predition of, linil responses is demonstrted when mesuring the virl response t Week. The odds rtio lultions re undefined when NPV is 100% or PPV is 100% or missing. Where the denomintors for oth the NPV nd PPV re greter thn five, minimum odds rtio ws determined y sutrting one speimen from the numertor of the 100% prmeter estimte (NPV or PPV). HBeAg-Negtive Ptients Chrteriztion of Virl Lod Tle 32 elow demonstrtes the effiy, sed on HBV virl lod testing, of treting HBeAg-negtive ptients with 10 mg defovir dipivoxil ompred to pleo. At Week,.72% (57/117) of HBeAg-negtive ptients on tretment versus 0% (0/55) on pleo hd hieved very low virl lods elow 100 IU/mL. Furthermore, 3.42% (4/117) of ptients on tretment versus 30.91 % (17/55) on pleo hd virl lods greter thn or equl to 10 6 IU/mL. Tle 32 Distriution of HBV Virl Lod t Week for HBeAg-Negtive Ptients Virl Lod Adefovir Dipivoxil Pleo (IU/mL) N % Cumultive % N % Cumultive % TND 9 7.69 7.69 0 0.00 0.00 < 15 20 17.09.79 0 0.00 0.00 15 - < 100 28 23.93.72 0 0.00 0.00 100 - < 10 3 33 28.21 76.92 7.73.73 10 3 - < 10 4 9 7.69 84.62 5 9.09 21.82 10 4 - < 10 5 6 5.13 89.74 11 20.00 41.82 10 5 - < 10 6 8 6.84 96.58 15 27.27 69.09 10 6 - < 10 9 4 3.42 100.00 17 30.91 100.00 10 9 0 0.00 100.00 0 0.00 100.00 Totl 117 100.00 55 100.00 TND = Trget Not Deteted 20