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1 Clin Chem Lb Med 2017; 55(4): Open Access Evelyn Stelzl, Hnnh M. Appel, Rochk Meht, Ed G. Mrins, Jörg Berg, Christin Pr, Hnn Zurl, Brigitte I. Sntner nd Hrld H. Kessler* Evlution of the new cobs HCV genotyping test bsed on rel-time PCRs of three different HCV genome regions DOI /cclm Received July 12, 2016; ccepted September 13, 2016; previously published online October 14, 2016 Abstrct Bckground: Determintion of the heptitis C virus (HCV) genotype nd discrimintion between HCV subtypes 1 nd 1b is still mndtory prior to nti-hcv tretment initition. The im of this study ws to evlute the performnce of the recently introduced cobs ssy (Roche) nd to compre it to two comprtor ssys. Methods: The cobs ssy is bsed on primerspecific rel-time polymerse chin rection (PCR). For comprison, the TRUGENE HCV 5 NC (Siemens) nd the VERSANT HCV Genotype 2.0 Assy (Siemens) were employed. Accurcy of the new ssy ws determined using proficiency pnels. For clinicl evlution, 183 residul clinicl smples obtined from ptients with chronic heptitis C infection were included. Results: When ccurcy ws tested, pnel members contining HCV subtypes 1, 1b, nd 3 were identified s expected; however, the new ssy filed to identify low titer pnel members contining HCV subtype 5 correctly. Of 183 clinicl smples, 160 gve concordnt results. For seven smples, n indeterminte result ws reported with the cobs ssy nd the remining 16 smples were found discordnt with one of the comprtor ssys. When time-to-results of the ssys were compred, the *Corresponding uthor: Hrld H. Kessler, Institute of Hygiene, Microbiology nd Environmentl Medicine, Medicl University of Grz, Universitätspltz 4, 8010 Grz, Austri, Phone: , Fx: , E-mil: hrld.kessler@medunigrz.t Evelyn Stelzl, Hnnh M. Appel, Hnn Zurl nd Brigitte I. Sntner: Institute of Hygiene, Microbiology nd Environmentl Medicine, Medicl University of Grz, Grz, Austri Rochk Meht nd Ed G. Mrins: Roche Moleculr Dignostics, Plesnton, CA, USA Jörg Berg nd Christin Pr: Institute of Lbortory Medicine, Kepler Universitätsklinikum, Medicl Cmpus III, Linz, Austri new ssy showed shorter totl time nd similr hndson time per smple. Conclusions: The cobs ssy showed good performnce nd proved to be suitble for use in the routine dignostic lbortory. Due to the high level of utomtion, fst nd relible results re obtined with short hnds-on time. Keywords: cobs ; genotype; heptitis C virus (HCV); rel-time PCR; subtype. Introduction Testing for the heptitis C virus (HCV) genotype hs been importnt in understnding HCV clssifiction, epidemiology, evolution, trnsmission clustering, tretment response nd nturl history [1]. According to the ltest version of the Europen Assocition for the Study of the Liver (EASL) guideline, the HCV genotype/subtype should be determined with n ssy tht ccurtely discrimintes HCV subtype 1 from 1b [2]. For determintion of the HCV genotype/subtype, different moleculr techniques my be used including popultion sequencing, reverse hybridiztion, nd the newly-developed primer-specific rel-time polymerse chin rection (PCR) [3, 4]. The new cobs ssy (Roche Moleculr Systems, Plesnton, CA, USA) bsed on primer-specific rel-time PCR (RT-PCR) ws brought on the mrket recently. This ssy is bsed on nucleic cid mplifiction for identifiction of HCV genotypes 1 to 6 nd subtypes 1 nd 1b using the cobs 4800 system. This system consists of two instruments: the cobs x 480 instrument which is used for utomted specimen processing followed by utomted mplifiction nd detection on the cobs z 480 nlyzer. The ssy includes genotype nd subtype specific primers for mplifiction of prts of the 5 UTR, Core, nd NS5B regions of the HCV genome nd fluorescent dye-lbeled oligonucleotide probes for differentition of 2016, Hrld H. Kessler et l., published by De Gruyter. This work is licensed under the Cretive Commons Attribution-NonCommercil-NoDerivtives 3.0 License.
2 518 Stelzl et l.: HCV genotyping bsed on rel-time PCR HCV geno- nd subtypes. Additionlly, n internl control monitors the smple preprtion nd RT-PCR process. The im of this study ws to evlute the performnce of the new cobs ssy nd to compre results with those obtined by the VERSANT HCV Genotype 2.0 Assy (LiPA) (Siemens Helthcre Dignostics Inc., Trrytown, NY, USA) nd the TRUGENE HCV 5 NC Genotyping Kit (Siemens). Mterils nd methods Moleculr ssys Chrcteristics of HCV genotyping ssys used in this study re shown in Tble 1. Briefly, the cobs (Roche) ws performed ccording to the mnufcturer s pckge insert instructions. This HCV genotyping test is for use on the cobs 4800 system. After utomted specimen processing, ech smple is mplified in three RT-PCR rections using genotype nd subtype specific primers nd fluorescent dye-lbeled oligonucleotide probes. The probes re lbeled with four different fluorescent reporter dyes, llowing simultneous detection of HCV nd up to three genotypes or subtypes in ech rection. The cobs ssy hs been in vitro dignostics (IVD)/Conformité Europeéne (CE)-lbeled but not FDA pproved. The TRUGENE HCV 5 NC (Siemens) nd the VER- SANT HCV Genotype 2.0 Assy (Siemens) were performed ccording to the mnufcturer s pckge insert instructions. For both of the ssys, HCV RNA ws extrcted using the generic protocol of the NucliSENS esymag instrument (biomérieux, Mrcy-l Etoile, Frnce) with n input volume of 500 μl nd n elution volume of 50 μl. For resolution of discrepnt results, sequencing of the HCV NS5B region ws done s described erlier [5]. Plsm smples (1000 μl) were extrcted on the MgNA Pure LC 2.0 (Roche Dignostics, Penzberg, Germny) using the MgNA Pure LC totl NA Isoltion Kit LV (Roche) ccording to the mnufcturer s instructions. An elution volume of 50 μl ws used. Reverse trnscription (RT) ws crried out from 3 μl smple elute by using Superscript III enzyme (invitrogen, Thermo Fisher Scientific, Wlthm, MA, USA) ccording to mnufcturer s instructions. The primer (finl concentrtion of 2 μm) for RT ws 5 -CTCAGGCCTAT-TGGCCTGGAG-3. Therefter, PCR from 10 μl of the RT product ws performed with GoTq Flexi DNA Polymerse (Promeg, Fitchburg, WI, USA) ccording to mnufcturer s instructions with the following primers: HCV-PR3: 5 - TAT GAY ACC CGC TGY TTT GAC TC -3 nd HCV-PR5 5 -GCN GAR TAY CTV GTC ATA GCC TC -3 t 0.4 μm finl concentrtion ech. Cycling ws initited with 95 C for 2 min followed by 10 s t 95 C, 30 s t 58 C, nd 30 s t 72 C, nd finl extension t 72 C for 5 min. Amplifiction products were purified with ExoSAP-IT (ffymetrix, Snt Clr, CA, USA) regent nd, then sequenced using the BigDye Termintor v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, Wlthm, MA, USA). Sequencing products were seprted on 3130 ABI sequencing unit (Applied Biosystems) ccording to mnufcturer s instructions. Forwrd nd reverse sequences were ligned nd nlyzed with the SeqScpe v2.6 nlysis softwre (Applied Biosystems). HCV genotypes were determined utilizing the online nlysis tool geno2pheno ( Study design The ccurcy of the cobs ws determined utilizing the Qulity Control for Moleculr Dignostics (QCMD) 2014 nd 2015 Heptitis C Virus Genotyping EQA progrm pnels ( Both pnels consisted of seven plsm smples contining different genotypes of HCV with HCV RNA concentrtions exceeding 1000 IU/ ml nd one smple negtive for HCV RNA. For evlution of the clinicl performnce, 183 consecutive residul serum smples obtined from ptients with chronic HCV infection were included. Smples hd been nlyzed in the routine dignostic lbortory utilizing the TRUGENE HCV 5 NC Genotyping Kit. The number of smples contining HCV genotypes 2, 3, nd 4 s well s subtype 1 nd 1b ws virtully equl. Furthermore, one smple contining HCV genotype 6 ws included. All results obtined with the cobs were compred with those obtined by the comprtor ssys. If smple reveled discordnce regrding genotype or subtype 1/1b, this smple ws investigted dditionlly by home-brew NS5B sequencing. Results When eight members of the QCMD 2014 Heptitis C Virus Genotyping EQA progrm were tested with the cobs, five of seven members positive for HCV RNA gve genotype nd subtype results s expected (Tble 2). Of two members contining HCV subtype 5, one ws clssified s HCV genotype 4 nd the other one tested indeterminte with the cobs. The HCV RNA negtive member Tble 1: Fetures of HCV genotyping ssys used in this study. Chrcteristics Mnufcturer Roche Moleculr Systems Siemens Helthcre Dignostics Siemens Helthcre Dignostics Home-brew ssy Assy nme Roche cobs Versnt HCV Genotype 2.0 Assy (LiPA) TRUGENE HCV 5 NC Home-brew NS5B sequencing Trget region(s) 5 UTR + core + NS5B 5 UTR + core 5 UTR NS5B Detection method Rel-time PCR Line probe ssy Sequencing Sequencing
3 Stelzl et l.: HCV genotyping bsed on rel-time PCR 519 Tble 2: Accurcy testing utilizing the Qulity Control for Moleculr Dignostics HCV Genotyping Proficiency Progrm Vil no. HCV RNA concentrtion, IU/mL HCV subgenotype expected HCV (sub)genotype obtined by the cobs E E b 1; 1b 3 Negtive Negtive Invlid E E b 1; 1b E Indeterminte b E E ; 1 HCV RNA not detected. b HCV RNA detected but no genotype or subtype identified. Tble 4: Resolution of indeterminte results. Smple no. HCV (sub)genotype result obtined by Roche cobs Versnt HCV Genotype 2.0 Assy (LiPA) 1 Indeterminte Indeterminte Indeterminte Interprettion not 3k possible 4 Indeterminte Indeterminte 4/c/d 4c 6 Indeterminte 4/c/d 4c 7 Indeterminte 6 6 HCV RNA detected but no genotype or subtype identified. TRUGENE HCV 5 NC Tble 5: Resolution of discordnt results. showed n invlid result indicting tht no HCV RNA ws detectble. The sme results were found when smples from the QCMD 2015 Heptitis C Virus Genotyping EQA progrm were nlyzed (Tble 3). When 183 clinicl smples were tested with the cobs nd results obtined were compred with those obtined with the TRUGENE HCV 5 NC Genotyping Kit nd the VERSANT HCV Genotype 2.0 Assy, 160 smples reveled concordnt results. Seven of 183 (3.8%) smples showed indeterminte results with the cobs indicting detection of HCV RNA without identifiction of the HCV genotype or subtype. Corresponding results obtined with comprtor ssys re shown in Tble 4. Virl lods of these smples rnged from 1.5E + 04 IU/mL to 1.4E + 06 IU/mL. Of 183 smples, 16 (8.7%) showed discordnt results with t lest one comprtor ssy (Tble 5). Seven smples were found to contin subtype 1 with the cobs but reveled Tble 3: Accurcy testing utilizing the Qulity Control for Moleculr Dignostics HCV Genotyping Proficiency Progrm Smple no. HCV (sub)genotype result obtined by Roche cobs Versnt HCV Genotype 2.0 Assy (LiPA) TRUGENE HCV 5 NC 1 1; 1 1 1b 1 2 1; 1 1 1b 1 3 1; 1 1 1b 1 4 1; 1 1 1b 1 5 1; 1 1 1b 1 6 1; 1 1 1b 1 7 1; 1 INP 1b 1 8 1; 1b 1b 1 1b b 1b 1b 11 1b; 2 2/c 2 1b 12 1b; 2 2/c 2 1b 13 1b; 2 2/c 2 1b 14 1b; 2 2/c 2 1b 15 1; 4 4e 4o 4o 16 1; 1; 1b INP, interprettion not possible. Homebrew NS5B sequencing Vil no. HCV RNA concentrtion, IU/mL HCV subgenotype expected HCV (sub)genotype obtined by the cobs 1 > 1.00E > 1.00E b 1; 1b 3 > 1.00E Negtive Negtive Invlid 5 > 1.00E > 1.00E ; 1 7 > 1.00E Indeterminte b 8 > 1.00E b 1; 1b HCV RNA not detected. b HCV RNA detected but no genotype or subtype identified. subtype 1b with one of the comprison ssys. Vice vers, one smple ws found to contin subtype 1b with the cobs but reveled genotyped 1 with one of the comprison ssys. Two smples reveled genotype 1 with the cobs but no subtype could be determined. In five smples, the cobs reported two different genotypes (1 nd 2 resp. 1 nd 4). For one smple, the cobs reported subtypes 1 nd 1b. Additionl results obtined by NS5B sequencing re shown in Tble 5. When single smple ws tested with the cobs HCV GT, the overll time required ws 200 min per smple
4 520 Stelzl et l.: HCV genotyping bsed on rel-time PCR Tble 6: Clcultion of times per single smple required for ssys used in this study. Times per smple required (Tble 6). The TRUGENE HCV 5 NC could be performed within 255 min. The totl time required for the VERSANT HCV Genotype 2.0 Assy ws 405 min. Hnds-on work ws found to be similr for ll of the ssys. Discussion Roche cobs Versnt HCV Genotype 2.0 Assy (LiPA) Assys TRUGENE HCV 5 NC Hnds-on time, min Totl time, min Determintion of the HCV genotype is prt of pre-therpeutic ssessment nd recommended with genotyping ssy tht determines genotypes nd discrimintes subtype 1 nd 1b ccurtely. The cobs is bsed on primer-specific rel-time PCR for identifiction of HCV genotypes 1 to 6 nd subtypes 1 nd 1b. In this study, the cobs ws evluted nd compred with the VERSANT HCV Genotype 2.0 Assy (LiPA) nd the TRUGENE HCV 5 NC. When members of the QCMD 2014 nd 2015 Heptitis C Virus Genotyping EQA progrm were tested, ll members except those with HCV genotype 5 were reported s expected. According to the pckge insert instructions of the cobs, the limit of detection for genotype 5 smples is 1000 IU/mL for plsm smples. For ll other genotypes, the limit of detection is 250 IU/mL or even lower. Virl concentrtions of the members included in the proficiency pnels were found to be very close to this limit. As low virl concentrtions re rrely found in the clinicl routine, it might be better to include smples with higher HCV concentrtions in the pnel. Further investigtions regrding HCV genotype 5 could not be performed becuse smples from ptients with HCV genotype 5 were not vilble. In Europe, the prevlence of HCV genotype 5 is extremely low; HCV genotype 1 is predominnt followed by HCV genotypes 2 nd 3 [6]. When clinicl smples were tested with the cobs, 160/183 (87.4%) showed corresponding results with the comprtor ssys bsed on genotypes nd subtypes 1 nd 1b. Indeterminte results with the cobs were found in 7/183 (3.8%) of smples indicting detection of HCV RNA without identifiction of the HCV genotype or subtype tht my be cused by oligonucleotide mismtches in the trget region. The percentge observed in the present study ws found to be similr to tht reported with nother commercilly vilble ssy bsed on rel-time PCR [7]. Of the remining 16 smples, seven smples were found to contin subtype 1 with the cobs but reveled subtype 1b with one of the comprtor ssys. Additionlly, one smple ws found to contin subtype 1b with the cobs but reveled genotyped 1 with one of the comprtor ssys. For these eight smples, results reported by NS5B sequencing confirmed results reported by the cobs. Two smples reveled genotype 1 with the cobs but the ssy filed to determine the subtype probbly due to oligonucleotide mismtches in the trget region. In four smples, the cobs reported genotypes 1 nd 2. When nlyzing these smples with the comprtor ssys nd NS5B sequencing, different results were found. HCV genotype 2 ws reported with both the VERSANT HCV Genotype 2.0 Assy (LiPA) using prts of the 5 UTR nd core regions nd the TRUGENE HCV 5 NC using the 5 UTR only. In contrst, NS5B sequencing reported genotype 1. Bsed on these results, the presence of recombinnt HCV strin in these ptients my be possible explntion. Severl reports bout HCV recombinnts hve been published from 2002 onwrds [8, 9]. For one smple, the cobs reported mixed infection with HCV subtypes 1 nd 1b. Another smple ws found to contin HCV genotypes 1 nd 4. When compring these results with those of the comprtor ssys, one subtype or genotype ech could be found with the sme genotype being reported by NS5B sequencing. These discordnt results my be explined by second HCV strin present in the ptient but with lower virl concentrtion. For bulk sequencing, it hs been known tht strins with proportion below 20% cnnot be detected. Next-genertion sequencing (NGS) my be superior lterntive; however, NGS protocols re currently not pproprite for the routine dignostic lbortory becuse they lck stndrdiztion. Furthermore, they re time-consuming, lbor- nd cost-intensive. Comprison of times required for the cobs with those required for the VERSANT HCV Genotype 2.0 Assy (LiPA) nd the TRUGENE HCV 5 NC Genotyping Kit reveled the shortest totl time for the cobs HCV
5 Stelzl et l.: HCV genotyping bsed on rel-time PCR 521 GT. However, hnds-on time ws similr for ll of the ssys. In conclusion, the new cobs ssy showed good performnce. It proved to be suitble for use in the routine dignostic lbortory. The new ssy llows correct determintion of the HCV genotypes 1 to 4 nd is ble to discriminte HCV subtype 1 from 1b. Due to the high level of utomtion, fst nd relible results re obtined with short hnds-on time. While the comprtor ssys need subjective interprettion of results, the new ssy provides utomted interprettion nd connection to the lbortory informtion system. Acknowledgments: The uthors grtefully cknowledge Dniel Wllner for technicl ssistnce. Author contributions: All uthors hve ccepted responsibility for the entire content of this submitted mnuscript nd pproved submission. Reserch funding: Prt of the regents required for this study were supported by Roche Moleculr Dignostics. Employment or ledership: None declred. Honorrium: None declred. Competing interests: The funding orgniztion plyed no role in the study design; in the collection, nlysis, nd interprettion of dt; in the writing of the report; or in the decision to submit the report for publiction. References 1. Simmonds P. Genetic diversity nd evolution of heptitis C virus 15 yers on. J Gen Virol 2004;85: Europen Assocition for the Study of the Liver. EASL recommendtions on tretment of heptitis C J Heptol 2015;63: Stelzl E, vn der Meer C, Gouw R, Beld M, Grhovc M, Kessler HH, et l. Determintion of the heptitis C virus subtype: comprison of sequencing nd reverse hybridiztion ssys. Clin Chem Lb Med 2007;45: Hoffmnn D, Michler T, Protzer U. Heptitis viruses. In: Kessler HH, editor. Moleculr dignostics of infectious diseses, 3rd ed. Berlin, Boston: De Gruyter, 2014: Morice Y, Roulot D, Grndo V, Stirnemnn J, Gult E, Jentils V, et l. Phylogenetic nlyses confirm the high prevlence of heptitis C virus (HCV) type 4 in the Seine-Sint-Denis district (Frnce) nd indicte seven different HCV-4 subtypes linked to two different epidemiologicl ptterns. J Gen Virol 2001;82: Mnns MP, von Hhn T. Novel therpies for heptitis C one pill fits ll? Nt Rev Drug Discov 2013;12: McCormick AL, Mcrtney MJ, Abdi-Abshir I, Lbbett W, Smith C, Irish D, et l. Evlution of sequencing of HCV core/e1, NS5A nd NS5B s genotype predictive tool in comprison with commercil ssys trgeting 5 UTR. J Clin Virol 2015;66: Gonzlez-Cndels F, Lopez-Lbrdor FX, Brcho MA. Recombintion in heptitis C virus. Viruses 2011;3: Klinin O, Norder H, Mukomolov S, Mgnius LO. A nturl intergenotypic recombinnt of heptitis C virus identified in St. Petersburg. J Virol 2002;76:
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