Published Ahead of Print 26 June 2013. 10.1128/JCM.00936-13.

2013, 51(9):2893. DOI:J. Clin. Microbiol. Karamitros, Alexis Vassilakis and Angelos HatzakisManolakopoulos, Ioannis Elefsiniotis, TimokratisEmanuel Manesis, George Papatheodoridis, Spilios Fotinie Ntziora, Dimitrios Paraskevis, Catherine Haida, Era of Personalized MedicineHepatitis B Virus-Resistant Strains in the RT-PCR) Assay for Detection of MinorityMutation System Real-Time PCR (ARMS Ultrasensitive Amplification Refractory

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Ultrasensitive Amplification Refractory Mutation System Real-TimePCR (ARMS RT-PCR) Assay for Detection of Minority Hepatitis BVirus-Resistant Strains in the Era of Personalized Medicine

Fotinie Ntziora,a Dimitrios Paraskevis,a Catherine Haida,a Emanuel Manesis,b George Papatheodoridis,c Spilios Manolakopoulos,c

Ioannis Elefsiniotis,d Timokratis Karamitros,a Alexis Vassilakis,a Angelos Hatzakisa

Department of Hygiene, Epidemiology, and Medical Statistics, University of Athens Medical School, Athens, Greecea; Division of Internal Medicine, University of AthensMedical School, Athens, Greeceb; Second Department of Internal Medicine, Hippokration General Hospital, Athens, Greecec; Hepatology Unit, Elena Venizelou Hospital,Athens, Greeced

Resistance to antiviral treatment for chronic hepatitis B virus (HBV) has been associated with mutations in the HBV polymeraseregion. This study aimed at developing an ultrasensitive method for quantifying viral populations with all major HBV resis-tance-associated mutations, combining the amplification refractory mutation system real-time PCR (ARMS RT-PCR) with a mo-lecular beacon using a LightCycler. The discriminatory ability of this method, the ARMS RT-PCR with molecular beacon assay,was 0.01 to 0.25% for the different HBV resistance-associated mutations, as determined by laboratory-synthesized wild-type(WT) and mutant (Mut) target sequences. The assay showed 100% sensitivity for the detection of mutant variants A181V, T184A,and N236T in samples from 41 chronically HBV-infected patients under antiviral therapy who had developed resistance-associ-ated mutations detected by direct PCR Sanger sequencing. The ratio of mutant to wild-type viral populations (the Mut/WT ra-tio) was >1% in 38 (63.3%) of 60 samples from chronically HBV-infected nucleos(t)ide analogue-naive patients; combinationsof mutations were also detected in half of these samples. The ARMS RT-PCR with molecular beacon assay achieved high sensitiv-ity and discriminatory ability compared to the gold standard of direct PCR Sanger sequencing in identifying resistant viral popu-lations in chronically HBV-infected patients receiving antiviral therapy. Apart from the dominant clones, other quasispecieswere also quantified. In samples from chronically HBV-infected nucleos(t)ide analogue-naive patients, the assay proved to be auseful tool in detecting minor variant populations before the initiation of the treatment. These observations need further evalua-tion with prospective studies before they can be implemented in daily practice.

Nucleos(t)ide analogues are a fundamental tool for the treat-ment of hepatitis B virus (HBV)-associated liver disease

because of their potent antiviral activity in the absence of re-markable side effects and major contraindications (1). Long-term nucleos(t)ide analog therapy among patients significantlyimproves survival and reduces the risk of liver-related major com-plications, such as death, hepatocellular carcinoma (HCC), andliver decompensation (2). A major concern with nucleos(t)ideanalogue therapy is the risk for the development of viral popula-tions with resistance-associated mutations (3, 4). Standard directPCR Sanger sequencing and point mutation assays are able todetect viral populations with resistance-associated mutations onlywhen they become dominant, leaving a gap in the understandingof the dynamics in the development of resistance. Direct PCRSanger sequencing detects, on average, mutations present at ratiosof �20% of the circulating virus population (5). Clonal sequenc-ing has a higher sensitivity for detecting low-prevalence HBV mu-tations, but it is costly and labor-intensive (6). Point mutationassays can detect specific variants at as low as 5% of the viruspopulation, but only if well-established mutations are present,whereas it may miss mutations that are not yet associated withresistance (2). Other sequence-specific genotypic resistance testsare available or under development, such as restriction fragmentlength/mass polymorphism, mutation-specific real-time PCR,oligonucleotide microarray, and gene chip technology (7, 8). Theuse of novel technologies to sequence multiple genetic variants ina heterogeneous pool of amplified DNA molecules, such as thosefrom a virus quasispecies, by massively parallelizing PCR amplifi-

cation, represents the next-generation sequencing (NGS) that isable to quantitatively detect minority variants at levels as low as 1to 2% (9, 10).

Our aim here was to develop a highly sensitive and reproduciblemethod for quantifying viral populations with all major HBV resis-tance-associated mutations combining the amplification refractorymutation system real-time PCR (ARMS RT-PCR) with molecularbeacon biotechnology, even if they represent minor quasispecies.

MATERIALS AND METHODSDNA extraction. Viral DNA was extracted from 500 �l of serum by usinga QIAamp DNA blood minikit (Qiagen NV, Netherlands) according tothe standard protocol. The extracted products from the clinical sampleswere stored at �80°C.

Primers and molecular beacon. A total of 67 HBV full-length se-quences representing all available human HBV genotypes (A to H) weredownloaded from the GenBank sequence database (http://www.ncbi.nlm.nih.gov) and aligned using the CLUSTAL W program (version 1.81) (11).

Received 9 April 2013 Returned for modification 6 May 2013Accepted 19 June 2013

Published ahead of print 26 June 2013

Address correspondence to Dimitrios Paraskevis, dparask@med.uoa.gr.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00936-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JCM.00936-13

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The most common point mutation was selected, especially regarding ge-notype D, for each one of the studied resistance-associated mutations.Primers were designed according to the ARMS technique so as to be re-fractory for the PCR amplification of the nonmatching target sequences(12–14). A series of experiments where mutant primers with four differentn�1 substitutions have been used under the same conditions of temper-ature and time allowed us to choose the best set of primers that gavesufficient discriminatory ability without decreasing the sensitivity andspecificity of the method. In cases where neighboring mutations werepresent, e.g., L180M, A181V, and T184A, we applied the same reverseprimer designed in a stable area of the targeted sequence and differentforward primers specifically designed for each mutation. In the positionswhere different bases might have been expected according to the align-ment of all possible combinations and expected variations attributed todifferent mutations, we inserted ambiguity codes in the forward primer’sdesign. The insertion of ambiguity codes did not affect the primer bind-ing, nor did it decrease the sensitivity of the ARMS RT-PCR with molec-ular beacon assay in any way. Single-stranded target sequence synthesisand high-purity clearance for primers was performed by Invitrogen Lab-oratories (Invitrogen Corp., Carlsbad, CA). The molecular beacon wassynthesized by Biolegio Laboratories (Biolegio BV, Netherlands). Theprimers and the molecular beacon we finally applied can be found in TableS1a in the supplemental material.

WT and Mut laboratory target sequences. A single-stranded DNA tar-get sequence containing 326 bases was designed as to include codons encod-ing for the wild-type (WT) virus amino acids at positions 173, 180, 181, 184,204, and 236. Based on the WT target sequence a mutant (Mut) target se-quence was constructed so that the most common point-mutational codonfor each mutation would be included. The laboratory designed target of 326bases was introduced to the plasmid by a standard cloning protocol. Thetarget was ligated into the pCR2.1 TOPO-TA vector. The ligated plasmidswere transformed into Escherichia coli XL-1 competent cells according to themanufacturer’s instructions (Invitrogen Corp., Carlsbad, CA). Transformedclones were selected using a blue-white selection system, and plasmids werepurified using a Miniprep DNA purification kit (TaKaRa Bio, Inc., Otsu,Japan). Insertion was verified by EcoRI digestion and sequencing (ABI3100Sequencer; Abbot Diagnostics, IL) using M13 primers. Plasmid-derived Mutand WT target sequences contained all six studied codons.

ARMS RT-PCR with molecular beacon reaction. Quant-iTPicoGreen double-stranded DNA reagent was applied to quantitate thePCR amplification product with real-time measurements using the Light-Cycler 2.0 system after the WT and Mut laboratory-designed target se-quences were converted to double-stranded DNA forms (Invitrogen andRoche Applied Science [Indianapolis, IN]). After the initial concentra-tions were calculated for both WT and Mut target sequences, appropriatedilutions were performed in order to obtain an initial concentration of 106

copies per reaction for each target. The LightCycler system was used forthe ARMS RT-PCR (Roche Diagnostics Corp., Roche Applied Science).

The final concentrations applied were as follows: 2 �l per reaction of 10�LC FastStart DNA Master HybProbe, 1 �l per reaction of each of theappropriate for the target sequence forward and reverse set of primersfrom a stock concentration of 30 �M, 5 �l per reaction of MgCl2 from astock concentration of 25 mM, 0.5 �l per reaction of the molecular beaconfrom a stock concentration of 20 �M, and 1 �l per reaction of heat-labileuracil-DNA glycosylase from a stock concentration of 1 U/�l. Next, 20-�lPCR capillary tubes were used for mixing 10.5 �l of Master Mix with 10 �lof the HBV DNA target sequences. ARMS RT-PCR conditions and differ-ent annealing temperatures applied are summarized in Table 1. Serialdilutions were used for WT and Mut target sequences, ranging from 106 to10 copies per reaction. The two sets of primers, one specifically designedfor the WT target and the other designed for the Mut target, were appliedto the relevant targets to construct a DNA standard curve. The overallworkflow of the ARMS RT-PCR with molecular beacon assay is shown inFig. 1. In early experiments, WT and Mut target sequences were mixed inpredetermined concentrations in order to resemble clinical samples.However, in the experiments performed in order to determine the dis-criminatory ability of the assay, WT and Mut target sequences were usedseparately with the appropriate sets of primers.

Evaluation of ARMS RT-PCR with molecular beacon assay in clini-cal samples. Based on our previous study, the biological cutoff the ARMSRT-PCR assay has been determined to be 0.04%, meaning that the assaywas able to detect the viral subpopulation in a clinical sample if it wasrepresented in at least 4 copies per 104 copies of the total viral population(14). For this reason, we decided that most of our clinical samples shouldhave a viral load �104 copies per reaction after dilution in order to beincluded in our study. The reference method for the viral load of HBV inthe patient samples was a method developed in-house (15). A total of 41plasma or serum samples from chronically HBV-infected patients whofailed antiviral treatment were selected for the evaluation of the ARMSRT-PCR with molecular beacon assay. Eligible patients included thosethat had shown antiviral resistance, proved by detectable HBV DNA intwo sequential measurements and the development of resistant mutationswhen tested by direct PCR Sanger sequencing. Sixty samples from chron-ically HBV-infected nucleos(t)ide analogue-naive patients were selectedfor measurement by the ARMS RT-PCR with molecular beacon assay. Theresults assessed the presence of resistant quasispecies in samples fromHBV-naive patients for each of the six mutational sites.

Bioethical approval. The Bioethical Committee of the MedicalSchool, Athens University, approved the conduct of the study under thetitle “Quantification of HBV Mutations Associated with Antiviral Resis-tance” (reference 37/26-4-06).

Statistical analysis. Constant variables were expressed as means, stan-dard deviations, medians, and percentiles. Pearson and Spearman corre-lation coefficients were applied for correlations between sequential vari-ables. McNemar’s test was used to assess discordance and differentiationfor the two methods. Sensitivity and 95% confidence intervals of the

TABLE 1 Final cycling conditions for each major HBV resistance-associated mutation

Reaction step

Final cycling conditions for indicated mutation

V173L L180M A181V T184A M204I �236�

Temp(°C)

Time(min)

Temp(°C)

Time(min)

Temp(°C)

Time(min)

Temp(°C)

Time(min)

Temp(°C)

Time(min)

Temp(°C)

Time(min)

Denaturation 94 10 94 10 94 10 94 10 94 10 94 1050 RT-PCR cycles

Initial temp 94 0 94 0 94 0 94 0 94 0 94 0Fluorescence measurement 52 0.02 52 0.02 52 0.02 52 0.02 52 0.02 52 0.02Annealinga 58 0.10 58 0.10 56 0.10 58 0.10 58 0.10 56 0.10

Final extension 72 0.10 72 0.10 72 0.10 72 0.10 72 0.10 72 0.10a The final cycling conditions applied for each of the major HBV resistance-associated mutations according to the ARMS RT-PCR with a molecular beacon assay are given.Different annealing temperatures were required for each mutation. The fluorescence measurement was performed in a different step of the reaction.

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ARMS RT-PCR with molecular beacon assay were calculated with the SAS9.0 (2004; SAS Institute, Inc.) statistical program (16).

RESULTS

ARMS RT-PCR with molecular beacon assay has been evaluatedusing serial dilutions of WT and Mut target sequences from 106

copies per reaction to 10 copies per reaction with the appropriateset of primers for every one of the HBV resistance-associated mu-tations (14). Repeated measurements showed that the assay suc-cessfully detected WT and Mut target sequences in concentrationsas low as 10 copies per reaction an analytical sensitivity of 90 to100% for each one of the studied mutations. Representative ex-amples of experiments performed in target sequences in order todetermine the discriminatory ability of the ARMS RT-PCR withmolecular beacon are shown in Fig. 2. The discriminatory abilityof the ARMS RT-PCR with molecular beacon assay was 0.01 to0.25% for the different HBV resistance-associated mutations, asshown in Table 2. Discriminatory ability has been evaluated inrepeated experiments (n � 5); however, measurements are basedon representative experiment data. The methodology of theARMS RT-PCR with molecular beacon assay focuses on the de-tection of Mut variants when they represent minor viral popula-tions. The experiments for the discriminatory ability were per-formed at the lowest concentrations for the WT (106 to 10variants) variants, suggesting that they apply also at higher values.The reaction has proven to be efficient to detect higher and lowerconcentrations of both Mut and WT targets, and the Mut/WTratio was kept constant for each of the tested mutations regardlessof the initial concentration of the dominant target sequence.

Evaluation of ARMS RT-PCR with molecular beacon assay insamples from chronically HBV-infected patients receiving anti-viral therapy. The ARMS RT-PCR with molecular beacon assay

was applied for the quantitative detection of Mut HBV variants in41 samples from chronically HBV-infected patients receiving an-tiviral therapy. The presence of HBV resistance-associated muta-tions was previously detected by Sanger sequencing. The ratio ofMut to WT viral populations in each sample (Mut/WT ratio) wasdetermined. The results from the quantitative detection of the WTHBV quasispecies were compared to the overall viral load for eachsample, and the correlations were statistically significant apartfrom the mutation N236T (see Table S2a in the supplementalmaterial). Measurements of each of the samples from the HBV-infected patients with the ARMS RT-PCR with molecular beaconassay were compared to the results obtained using direct PCRSanger sequencing. The ARMS RT-PCR with molecular beaconassay showed a sensitivity of 100% for the detection of mutantvariants A181V, T184A, and N236T. The sensitivity of the assayfor the remaining mutations was 75%, 96.3% and 93.3% for theV173L, L180M and M204I, respectively (Table 3). In most cases,the Mut/WT ratio tested by the ARMS RT-PCR assay was �99%when a resistance-associated mutation was determined by directPCR Sanger sequencing. The total number of mutations detectedby Sanger sequencing was 57. Except for three mutations in dif-ferent samples (mutations V173L, L180M, and �204) detectedby Sanger sequencing, the rest were quantitatively detected byARMS RT-PCR with a molecular beacon assay as dominant pop-ulations, leading to an overall sensitivity for the ARMS RT-PCRassay of 54 of 57 (94.7%), a sensitivity comparable to other avail-able ultrasensitive assays. However, the total number of mutationsquantitatively detected by the ARMS RT-PCR assay was 77, al-though not always as dominant populations. Upon reexaminationof the three samples, the viral load after the dilution was very lowwhen the samples were tested by ARMS RT-PCR (lower than thedescribed biological cutoff for the assay), whereas the samples had

FIG 1 ARMS RT-PCR with molecular beacon assay results. Each sample was quantitated twice; point k represents the sample quantification with the master mixcontaining the set of primers specifically designed to detect the WT sequence, and point l represents the sample quantification with the master mix containing theset of primers specifically designed to detect the Mut sequence. In the final graph, the x axis represents the sample concentration calculated by the ARMS RT-PCRwith molecular beacon reaction, while the y axis represents the cycle and the fluorescence channel on which the sample has been detected by the reaction. TheX2/X1 ratio represents the Mut/WT ratio applied in data analysis. The procedure was repeated six times for the six different mutations.

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been tested using sequencing in higher concentrations before be-ing diluted; therefore, the inability of the ARMS RT-PCR to detectthe Mut variants was due to the low concentration of HBV vari-ants following the dilution. Moreover, sequencing results for theV173L mutation gave a mixed population of V173V/L for thesample where ARMS RT-PCR failed to detect a Mut/WT ratio.Apart from the dominant viral populations, minor variant popu-lations were also detected with the ARMS RT-PCR with a molec-ular beacon assay that might have contributed to the overall resis-tance profile of each patient (see Table S3a in the supplementalmaterial).

Evaluation of ARMS RT-PCR with a molecular beacon insamples from chronically HBV-infected, nucleos(t)ide ana-logue-naive patients. ARMS RT-PCR with a molecular beaconwas used to quantify Mut HBV variants in 60 samples from chron-ically HBV-infected, nucleos(t)ide analogue-naive patients. The

TABLE 2 ARMS RT-PCR discriminatory ability and cycle difference foreach HBV resistance-associated mutation

MutationDiscriminatoryabilitya (%)

Cycle difference(no. of differences)

V173L 0.010 9L180M 0.010 16A181V 0.250 11T184A 0.125 8M204I 0.125 16N236T 0.125 9a The discriminatory ability and cycle difference of the ARMS RT-PCR with a molecularbeacon assay for each of the HBV resistance-associated mutations (V173L, L180M,A181V, T184A, M204I, and N236T) is presented. Discriminatory ability was defined asthe lowest detected Mut variant concentration within the window of the assayperformed when WT and Mut primers were applied on the WT target sequence.

FIG 2 Representative results of experiments performed in target sequences in order to determine the discriminatory ability of the ARMS RT-PCR with molecularbeacon assay. The discriminatory ability represents the ratio (%) of the lowest Mut target sequence concentration that can be detected with the ARMS RT-PCRwith molecular beacon assay without interference by the WT target sequence using Mut primers divided by the WT target concentration. It was operationallydefined as the lowest detected Mut variant concentration within the window of the assay performed when WT and Mut primers were applied to the WT targetsequence. In the example of the resistance-associated mutation V173L (A), there is a nine-cycle difference window between the matching and nonmatchingprimers which enables the quantitative detection of Mut target sequences with concentrations of 103, 5 � 102, and 102 copies per reaction with the set of primersspecifically designed for the detection of the Mut target sequence (blue lines). However, a Mut target sequence concentration of 10 copies per reaction cannot bedetected with the matching set of primers within the red-line window, yielding a discriminatory ability of 0.01% for this mutation. In the example of theresistance-associated mutation A181V (B), blue lines represent the detection of Mut target sequences in concentrations 105, 104, 103, and 2.5 � 103copies perreaction with the matching Mut set of primers within the 11-cycle difference window between the matching and nonmatching primers for the WT targetsequence, yielding a discriminatory ability of 0.25% for this mutation.

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ratio of Mut to WT viral populations in each sample (i.e., theMut/WT ratio) was also determined. The results for the WT HBVquasispecies were compared to overall viral load for each sampleand the log HBV DNA (as calculated by the HBV DNA real-timePCR standard protocol) showed a good correlation with the logWT (as calculated by the ARMS RT-PCR with a molecular beaconassay) (see Table S4a in the supplemental material). The ratio ofmutant to wild-type viral populations in each sample (Mut/WTratio) was �1% in 38 (63.3%) of 60 samples from chronicallyHBV-infected, nucleos(t)ide analogue-naive patients. Combina-tions of resistance-associated mutations with a Mut/WT ratio�1% were detected in 65 of 209 (31%) measurements in which aMut/WT ratio could be estimated. In some cases, the Mut/WTratio was �99%. The results were categorized in the followinggroups: below the cutoff or a Mut/WT ratio of 1%, 1 to 5%, 5 to20%, or �20% for each one of the HBV resistance-associatedmutations, as shown in Table 4. The T184A mutation showed ahigher proportion of Mut/WT ratios that were �1% (22 samples),followed by N236T, A181V, L180M, and M204I (18, 15, 5, and 5samples, respectively), whereas no Mut viral population was de-tected over the predetermined ratio of 1% for V173L mutation.Interestingly, combinations of mutant quasispecies were found in19 (50%) of the 38 samples in which a Mut/WT ratio of �1% wasdetected. The most common combinations were T184A-N236Tand A181V-N236T (eight samples), followed by the combinationA181V-T184A (six samples). The percentage of mutant popula-tions was higher at L180, A181, and T184 resistant sites, probablydue to the high fitness cost of major resistance sites (e.g., M204 orN236) in the absence of therapy. Analytical results can be found inTable S5a in the supplemental material. We present there a com-parison of the results obtained with ARMS RT-PCR with a molec-ular beacon assay with two different thresholds— one at aMut/WT ratio of �1% and one at Mut/WT ratio of �5%—toshow the relative benefits in the quantitative detection of viralvariants with HBV resistance-associated mutations with an ultra-sensitive assay (Fig. 3).

DISCUSSION

Our data show that the ARMS RT-PCR with molecular beaconassay is an ultrasensitive method for the quantitative detection ofHBV quasispecies with nucleos(t)ide analogues resistance-associ-ated mutations before they emerge as dominant populations. Thisassay showed high sensitivity and discriminatory ability comparedto the direct PCR Sanger sequencing currently used in clinicalpractice in identifying resistant viral populations in chronicallyHBV-infected patients under antiviral therapy. The ARMS RT-PCR with molecular beacon assay yielded results comparable tothose of other novel ultrasensitive methods, such as next-genera-tion sequencing (NGS), for the quantitative detection of quasispe-cies, even in cases where combinations of resistance-associatedmutations exist. Moreover, ARMS RT-PCR provides a low-cost,user-friendly method that can be easily implemented in clinicallaboratories with no access to NGS technologies.

Studies in patients with HBV and/or HIV infection, both drug-naive and treated, showed that NGS application succeeded in de-tecting clinically relevant minority drug resistance mutants evenwhen they represented 1% of the total viral population (17, 18).The ARMS RT-PCR with molecular beacon assay quantitativelydetected, apart from the dominant clones, minor variant popula-tions, even when they represented 0.01 to 0.25% of the total viralpopulation, depending on the mutation tested. One of the majorlimitations of NGS technologies is the different types of errors thatcan be introduced during the PCR amplification step, such as thenucleotide misincorporation due to the inaccuracy of DNA poly-merases (19). In the ARMS RT-PCR assay, genome amplificationis not necessary since the quantification of specific variants is ac-complished due to differences in hybridization between the WTand Mut alleles and primers targeting specific positions. Thesecharacteristics may be the reason why the ARMS RT-PCR withmolecular beacon assay is more sensitive in the detection of viralquasispecies. Moreover, the ARMS RT-PCR with molecular bea-con assay can be easily adopted for the quantitative detection ofnovel or other well-established HBV resistance-associated muta-tions, with only slight modifications in the primers’ design and inthe annealing temperatures of the reaction.

Early detection of minor viral populations before they emergeto dominant clones may have an impact in the clinical outcome of

TABLE 3 Comparison of results obtained by ARMS RT-PCR with amolecular beacon assay and Sanger sequencing for HBV resistance-associated mutations in 41 samples from chronic HBV patients underantiviral therapy in whom HBV had developed resistance-associatedmutations

Mutation

No. of samples (n � 41)Sensitivitya (%)(95% CI)ARMS RT-PCR Sequencing

V173L 4 4 75b (19.4–94.4)L180M 30 27 96.3 (81–99.9)A181V 7 5 100 (47.8–100)T184A 9 2 100 (15.8–100)M204I 16 15 93.3 (68.1–99.8)N236T 11 4 100 (39.8–100)a Sensitivity was defined as the percentage of mutations detected by Sanger sequencingthat were also quantitatively detected by the ARMS RT-PCR assay. CI, confidenceinterval.b In the V173L mutation, although the ARMS RT-PCR assay and Sanger sequencingdetected the same numbers of positive samples, one sample that was detected by Sangersequencing was missed by the ARMS RT-PCR assay, whereas another sample testednegative for the V173L mutation with Sanger sequencing. ARMS RT-PCRquantitatively detected a viral subpopulation with the V173L mutation in a Mut/WTratio of 3.3%.

TABLE 4 Mut/WT ratio in 60 samples from chronically HBV-infectednucleos(t)ide analogue-naive patientsa

Mutation

No. of samples with the indicated Mut/WT ratio

Below thecutoff 1% 1–5% 5–20% �20%

V173L 49 11 0 0 0L180M 46 9 1 2 2A181V 18 27 5 4 6T184A 2 36 11 6 5M204I 31 24 0 0 5N236T 5 37 6 6 6a Quantitative detection of the Mut/WT ratio was performed in 60 samples obtainedfrom chronically HBV-infected nucleos(t)ide analogue-naive patients using ARMS RT-PCR with a molecular beacon assay. Each mutation was quantitatively detected withMut and WT primers, and the Mut/WT ratio was calculated. Results are grouped asfollows: a Mut/WT ratio below the cutoff or a Mut/WT ratio of 1%, 1 to 5%, 5 to20%, or �20% for each of the HBV resistance-associated mutation (V173L, L180M,A181V, T184A, M204I, and N236T).

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chronically HBV-infected patients. Several studies have been per-formed to determine whether mutational patterns of the HBVgenome are related to clinical outcomes after the emergence ofdrug-resistant variants during nucleos(t)ide analog therapy (20).Enomoto et al. (21) reported that an additional L180M mutationwas detected in 4 of the 9 patients who had the M204I mutationsalone at the time of first detection. Elevated alanine transaminase(ALT) activity was observed when the L180M mutation was de-tected in addition to the mutation M204I. The worst outcome wasattributed to the close interaction between position 180 in the173-189 helix and position 204 of the YMDD loop, as shown bythree-dimensional structural modeling (22). In the example ofadefovir dipivoxil, initially used for the treatment of patients withlamivudine resistance mutations, a more complex dynamic thanpreviously thought raised concerns regarding the clinical signifi-cance of early detection of minor viral quasispecies (23). Salvagetherapy in patients with lamivudine resistance achieves morerapid and higher rates of virological response if adefovir dipivoxilis initiated at an early phase of lamivudine resistance with lowlevels of viral replication, suggesting that early detection of mutantviral quasispecies using a sensitive technique may be crucial forpatients under long-term antiviral treatment in order to detectresistance and initiate salvage therapy promptly (24).

Sensitive HBV resistance assays may be useful even in the cur-rent era of entecavir and tenofovir, since there are still patientswho are not nucleos(t)ide naive but have been previously exposed

to other antiviral agents. The long-term efficacy of tenofovirmonotherapy for HBV-monoinfected patients after the failure ofnucleos(t)ide analogues has been clearly shown, but entecavirtherapy is associated with increasing rates of resistance (25, 26).The resistance rates to entecavir in lamivudine-experienced pa-tients are highest in cases with lamivudine resistance mutations atthe onset of entecavir, intermediate in patients with a history oflamivudine resistance, and relatively lower, but still much higherthan the entecavir resistance rates in naive patients, in patientswith exposure but no resistance to lamivudine, at least accordingto standard resistance assays (27). Novel sensitive HBV resistanceassays might be helpful in identifying lamivudine-exposed pa-tients with lamivudine-resistant HBV strains as minor variantpopulations, who will have an increased risk of developing resis-tance if they receive long-term entecavir monotherapy. Combina-tion therapy with entecavir and tenofovir has been used as salvagetherapy in chronically HBV-infected patients with multidrug viralresistance patterns or a partial antiviral response to precedingtherapies with promising results (28).

In conclusion, our results suggest that genotyping analysis,based on the next-generation ARMS RT-PCR with molecular bea-con assay, is suitable for characterization of genetic diversity anddetection of minor viral quasispecies that may influence therapeu-tic decisions in both nucleos(t)ide-naive and -experienced HBV-infected patients. The cost of the ARMS RT-PCR with molecularbeacon assay is low compared to other novel molecular tech-

FIG 3 Frequencies of observed combinations of mutant quasispecies in samples from HBV-naive patients in which the Mut/WT ratio was �1% when detectedby the ARMS RT-PCR with molecular beacon assay. The results are compared to those obtained with a Mut/WT ratio of �5%.

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niques, and future applications in clinical practice may lead toless-frequent HBV DNA level measurements and so reduce theoverall burden for the follow-up of chronically HBV-infected pa-tient under therapy. Moreover, this assay leads to a more person-alized approach, which is especially important for chronicallyHBV-infected patients since they are candidates for developingresistance-associated mutations and fail to be cured (29). Our datamay have a clinical significance when planning a treatment strat-egy in chronically HBV-infected patients in order to avoid thepotential development of HBV resistance-associated mutations;HBV viral quasispecies with resistance-associated mutations weredetected in a high proportion of our nucleos(t)ide-naive chroni-cally HBV-infected patients, suggesting that the initiation of inap-propriate therapy may trigger the emergence of viral resistance.Novel, sensitive, and accurate methods that enable the early de-tection of minor viral quasispecies before they emerge as domi-nant populations could lead to better targeted antiviral therapyand increased chances of success, but this hypothesis remains to beprospectively confirmed by clinical studies before it can be appliedto everyday practice.

ACKNOWLEDGMENTS

This study was supported in part by the Hellenic Scientific Society forResearch on AIDS and Sexually Transmitted Diseases and Gilead Hellas.This study was also supported by EASL young investigator full-bursaryawards.

F.N., D.P., and A.H. designed the study. F.N. and D.P. designed theARMS RT-PCR assay. F.N. performed the experiments and elaborated thedata. D.P. was consulted during the experiment’s progress. C.H., T.K.,and A.V. performed DNA extraction, HBV DNA testing, cloning experi-ments, and searches of the databases. E.M., G.P., S.M., and I.E. were re-sponsible for clinical follow-up. F.N. wrote the first draft of the manu-script. F.N., D.P., G.P., and A.H. edited all versions of the manuscript. Allauthors read, edited, and approved the final manuscript. We acknowledgethe assistance of BioMedes, Ltd., for English language editing.

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