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Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis Yi Mou,a,b Muhammad Ammar Athar,a,b Yuzhen Wu,a,b Ye Xu,a,b Jianhua Wu,c Zhenxing Xu,c Zulfiqar Hayder,d Saeed Khan,e Muhammad Idrees,f Muhammad Israr Nasir,g Yiqun Liao,a,h Qingge Lia,b State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, Chinaa; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, Chinab; Xiamen Hospital of Traditional Chinese Medicine, Xiamen, Fujian, Chinac; Department of Pathology, Quid-e-Azam Medical College, Bahawalpur, Punjab, Pakistand; Department of Molecular Pathology, Dow University of Health Sciences, Karachi, Pakistane; Center for Applied Molecular Biology, University of the Punjab, Lahore, Pakistanf; Department of Molecular Pathology, Liaquat National Hospital, Karachi, Pakistang; School of Public Health, Xiamen University, Xiamen, Fujian, Chinah
Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 104 copies/l. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries.
H
epatitis B is caused by the hepatitis B virus (HBV), an enveloped DNA virus that infects the liver, causing hepatocellular necrosis and inflammation (1). Chronic hepatitis B (CHB) infection affects approximately 248 million people worldwide and is a leading cause of liver-related morbidity and mortality, particularly in low- and middle-income countries (LMICs) (2). Patients with CHB can be successfully treated using nucleos(t)ide analogs (NAs), but drug-resistant HBV mutants frequently arise, leading to treatment failure and progression to liver disease (3). The development of drug resistance begins with mutations in the HBV polymerase gene, followed by an increase in the viral load and serum alanine aminotransferase levels several weeks to months later (4). Detection of drug resistance mutations is thus critical in prompt decision making for new therapeutic regimes (5, 6). A method enabling detection of NA resistance mutations should be reliable, rapid, and, in particular, easy to use and cost-effective when the aim is for it to be used in high-CHB-burden countries, which are often undeveloped and resource limited. Many methods for the detection of anti-HBV drug resistance mutations have been developed. However, these methods are almost exclusively performed by highly skilled technicians in wellequipped referral hospitals. This limitation restricts the use of anti-HBV drug resistance assays to only a small number of patients, which offers little help for CHB management program in LMICs. Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (referred to here as PCR Sanger sequencing)
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is currently the “gold standard” method for the detection of antiHBV drug resistance mutations. It is advantageous for the accurate detection of all nucleotide mutations, including novel mutations which have never been reported before. However, the Sanger sequencer is often unaffordable for the local hospitals that admit a majority of CHB patients in LMICs. Moreover, the sequencing procedure is complex and lengthy and requires very careful operations to avoid contamination from PCR amplicons. This is also true for other sequencing-based assays, such as pyrosequencing (7), coamplification at a lower denaturation temperature (COLD)-
Received 27 February 2016 Returned for modification 12 May 2016 Accepted 5 August 2016 Accepted manuscript posted online 17 August 2016 Citation Mou Y, Athar MA, Wu Y, Xu Y, Wu J, Xu Z, Hayder Z, Khan S, Idrees M, Nasir MI, Liao Y, Li Q. 2016. Detection of anti-hepatitis B virus drug resistance mutations based on multicolor melting curve analysis. J Clin Microbiol 54:2661–2668. doi:10.1128/JCM.00439-16. Editor: Y.-W. Tang, Memorial Sloan-Kettering Cancer Center Address correspondence to Yiqun Liao, [email protected], or Qingge Li, [email protected]. Y.M. and M.A.A. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00439-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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PCR Sanger sequencing (8–10), and next-generation sequencing (11, 12), as well as the high-end instrument-based assays, such as DNA microchip (13) and mass spectrometry (14) assays. Several simpler and more cost-effective assays for detecting anti-HBV drug resistance mutations have been developed. For example, the line probe assay (HBV DR v.3; Fujirebio Europe, Ghent, Belgium) is a reverse line blot assay that detects 27 mutations at 11 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The HBV DR v.3 assay is technically simple, rapid, and amenable to high throughput (15). However, as an open-tube assay, it requires multiple post-PCR hybridization and visualization steps, which are prone to amplicon contamination, which leads to the generation of false-positive results. Real-time PCR is an excellent platform of choice for clinical diagnosis because of its closed-tube detection format, which makes it easy to use and rapid and which results in decreased amplicon contamination. A realtime amplification refractory mutation system (ARMS) PCR for the detection of six HBV resistance mutations has been developed. Nevertheless, it is far from practical due to the limited coverage of mutation types (16). We previously reported a multiplex real-time PCR detection strategy based on multicolor melting curve analysis (MMCA) using dually labeled, self-quenched probes (17). Up to 16 genetic mutations could be detected in a single reaction by this strategy (18). In the present study, we describe a two-reaction MMCA assay that can detect 24 anti-HBV drug resistance mutations at 10 amino acid positions. We systematically evaluated its analytical performance, including its mutation detection accuracy, its analytical sensitivity, and, in particular, its ability to detect minor variants. We further evaluated its clinical performance by analyzing samples from a group of 212 CHB patients who had received nucleos(t)ide analog treatment, samples from a second group of 276 HBV-positive people who were selected from among those receiving a routine health examination, and a third group of 400 HBV-positive samples collected from individuals in Pakistan. The results were compared with those of PCR Sanger sequencing and confirmed by COLD-PCR Sanger sequencing. MATERIALS AND METHODS Clinical samples. In total, 888 serum samples were collected from the same number of deidentified patients. These samples were labeled HBV positive when they were received. The clinical samples were divided into three groups. Samples in group A (212 samples) were collected from patients who had received or were undergoing treatment with NAs at the Xiamen Hospital of Traditional Chinese Medicine (Xiamen, China). Samples in group B (276 samples) were collected in the same hospital from individuals who were being seen for a routine health examination and had never received NA drugs. Samples in group C (400 samples) were collected from four medical colleges or hospitals in Pakistan, including Quid-e-Azam Medical College, Dow University of Health Sciences, University of the Punjab, and Liaquat National Hospital. An exemption from full committee review for human subject studies was granted by the Xiamen University Research Ethics Committee since only coded samples collected for other procedures were used in the present work. HBV DNA was extracted from the serum samples using a Lab-Aid 820 virus DNA isolation kit (Zeesan Biotech Ltd., Xiamen, China). The DNA extracts were used for both MMCA and PCR Sanger sequencing. Before mutation detection, the presence of HBV in the sample was confirmed by use of an HBV real-time PCR quantification kit (Zhijiang Biotech Ltd., Shanghai, China). Plasmid DNA. Wild-type plasmid DNA was prepared by use of an HBV-positive sample obtained from the Xiamen Hospital of Tradi-
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tional Chinese Medicine. An amplicon of 828 bp from reverse transcriptase amino acid 14 (rt14) to rt289 was generated using forward primer 5=-ATCAGGACTCCTAGGACC-3= and reverse primer 5=-TCGTTGACA TACTTTCCAATC-3=. Sequencing analysis confirmed that the amplicon corresponded to that from the HBV isolate with GenBank accession number GQ377639.1 (http://www.ncbi.nlm.nih.gov/) with one non-drug resistance mutation (A ¡ G) on the third base of rt255. A plasmid carrying the wild-type HBV sequence was constructed by cloning the amplicon into the pMD18-T vector using a TA cloning kit (TaKaRa, Dalian, China). Plasmids carrying mutant DNA sequences were constructed from the wild-type plasmid by site-directed mutagenesis (19). In total, 24 plasmids with 24 mutations, i.e., rtL80I, rtL80V, rtV173L, rtV173G, rt(L/V)180M, rtA181T, rtA181V, rtT184A, rtT184C, rtT184F, rtT184G, rtT184I, rtT184L, rtT184M, rtT184S, rtA194T, rtS202C, rtS202G, rtS202I, rtM204I, rtM204V, rtM204S, rtI233V, and rtN236T, were prepared. The drug resistance nucleotide mutation(s) corresponding to each amino acid alteration was determined (see Table S1 in the supplemental material). The plasmid DNA in 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA (TE buffer) was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE). MMCA assay. The MMCA assay has two reactions that detect 24 mutations in HBV that confer resistance against the five FDA-approved antiHBV NA drugs, including lamivudine (LMV), adefovir (ADV), entecavir (ETV), tenofovir (TDF), and telbivudine (LdT). Reaction A detects 10 mutations, including rtL80I, rtL80V, rt(L/V)180M, rtA181T, rtA181V, rtA194T, rtM204I, rtM204V, rtM204S, and rtN236T. Reaction B detects 14 mutations, including rtV173L, rtV173G, rtT184A, rtT184C, rtT184F, rtT184G, rtT184I, rtT184L, rtT184M, rtT184S, rtS202C, rtS202G, rtS202I, and rtI233V. Each MMCA assay reaction mixture contains 2.5 l of 10⫻ Taq HS buffer (TaKaRa), 0.2 l of Taq HS (5 units/l), 0.2 l of deoxynucleoside triphosphates (dNTPs; 25 mM), 0.1 l of primer-probe mix, 17 l of deionized H2O, and 5 l of the HBV DNA template. PCR and melting curve analysis were performed in a Bio-Rad CFX-96 thermocycler (BioRad, Hercules, CA). PCR was started with denaturation at 95°C for 5 min and was followed by 45 cycles of 95°C for 15 s, 52°C for 15 s, and 72°C for 20 s. Melting curve analysis began with denaturation at 95°C for 60 s and hybridization at 30°C for 60 s, followed by a gradual temperature increase from 30°C to 85°C in increments of 0.5°C/s. The fluorescence from the 6-carboxyfluorescein (FAM; absorbance [abs] and emission [em], 495 nm and 520 nm, respectively), hexachloro-6-carboxyfluorescein (HEX; abs and em, 521 nm and 536 nm, respectively), 6-carboxy-Xrhodamine (ROX; abs and em, 586 nm and 610 nm, respectively), and indodicarbocyanine 5 (Cy5; abs and em, 647 nm and 667 nm, respectively) channels was recorded. PCR and COLD-PCR for Sanger sequencing. Each 25-l PCR mixture was prepared as described above and contained 2.5 l of 10⫻ Taq HS buffer, 0.2 l of Taq HS, 0.2 l of dNTPs, 0.1 l of primer mix, 17 l of deionized H2O, and 5 l of HBV DNA. The primer mix was composed of 20 pM each forward primer 5=-ATCAGGACTCCTAGGACC-3= and reverse primer 5=-TCGTTGACATACTTTCCAATC-3=. Conventional PCR was carried out in a T3 thermocycler (Biometra, Göttingen, Germany) using a program of 95°C for 5 min, followed by 45 cycles of 95°C for 15 s, 52°C for 15 s, and 72°C for 40 s. COLD-PCR was performed essentially as previously described (10). Briefly, 5 l of the HBV DNA template was added to a 20-l PCR mixture containing 2.5 l of 10⫻ Taq HS buffer, 0.2 l of Taq HS (5 unit/l), 0.1 l of primer mix (50 M for each), 0.2 l of dNTPs (25 mM), and 17 l of deionized H2O. Amplification was carried out in a RotorGene Q realtime PCR system (Qiagen, Hilden, Germany) under the following conditions: 95°C for 10 min; 10 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 1 min; 72°C for 7 min; 95°C for 2 min; and finally, 30 cycles of 95°C for 15 s, 70°C for 1 min, 77°C for 5 s, 50°C for 30 s, and 72°C for 1 min. The amplified products were sent out for bidirectional Sanger sequencing (Sangon Inc., Shanghai, China).
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FIG 1 Readout for a wild-type plasmid and 24 mutant plasmids using the MMCA method. (Top) Typical readout for reaction A; (bottom) typical readout for reaction B. Black lines, melting curve of wild-type plasmid; gray lines, melting curves of 24 mutant plasmids. WT, wild type; ⫺dF/dt, negative derivative of fluorescence over temperature; TET, tetrachloro-6-carboxyfluorescein.
RESULTS
We evaluated the analytical performance of the MMCA assay by using a plasmid carrying wild-type HBV DNA (wild-type plasmid) and 24 plasmids carrying mutant HBV DNA (mutant plasmids) as mimics of the respective wild-type and mutant HBV DNA samples. For each plasmid, we prepared serial 10-fold dilutions in TE ranging from 106 to 100 copies/l. The MMCA assay showed that for each plasmid, a unique melting temperature (Tm) value could be obtained regardless of the concentration. Figure 1 shows the melting curves of all the plasmids at 10 copies/l. Ten repeated detections at this concentration provided the average Tm and standard deviation (SD) for each plasmid (Table 1). All the Tm values for the mutant plasmids had a ⬎4°C difference from the Tm value for the wild-type plasmid, and the SD values were less than 1°C. As the Tm resolution of the MMCA assay was 0.5°C, all mutant plasmids investigated could be unequivocally differentiated from the wild-type plasmid. The analytical sensitivity of the assay was obtained through 10 repeated detections of each plasmid at 10 copies/l and 1 copy/l. The results showed that all plasmids at 1 copy/l or 5 copies per reaction could be detected (Fig. 2), and the limit of detection of the MMCA assay was thus 1 copy/l, or 5 copies per reaction. To test the ability of MMCA assay to detect minor mutant subpopulations, we prepared a series of mixtures containing plasmids carrying the wild type and the rtM204V mutant. The overall template concentration was set to 104 copies/l, and the percentage of the population that was the rtM204V mutant was 0%, 5%, 10%, and 20%. These mixtures were subjected to MMCA, PCR Sanger sequencing, and COLD-PCR Sanger sequencing in parallel. The results showed that both the MMCA assay and COLDPCR Sanger sequencing could detect the rtM204V mutant subpopulation when it was present at proportions of 5% and above, whereas PCR Sanger sequencing method could detect the subpopulation when it was present at proportions of 10% and above
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(Fig. 3), demonstrating that MMCA has a sensitivity for the detection of minor mutant subpopulations equal to that of COLD-PCR Sanger sequencing, which is more sensitive than PCR Sanger sequencing. To evaluate the clinical performance of MMCA, we used three different groups of patient samples. The results showed that all samples in group A were confirmed to be HBV positive. The mutations detected are listed in Table 2. Of the 212 samples, 103 (48.6%) were found to contain anti-HBV drug resistance mutations, indicating the widespread prevalence of drug resistance in this group. An LMV monoresistance mutation (70.9%, 73/103) was the most frequently detected mutation, followed by an ADV monoresistance mutation (21.4%, 22/103), an LMV and ETV resistance mutation (4.9%, 5/103), and an LMV and ADV resistance mutation (2.9%, 3/103) (Fig. 4A). MMCA detected 6 more samples with mutations than PCR Sanger sequencing did, and these were found to carry minor mutant subpopulations that escaped detection by PCR Sanger sequencing. These results were confirmed by COLD-PCR Sanger sequencing. In group B, 274 samples (99.3%, 274/276) were confirmed to be HBV positive, and all of these gave MMCA results. Nine samples (3.3%, 9/274) were found to contain anti-HBV drug resistance mutations, including 7 LMV monoresistance mutations and 2 ADV monoresistance mutations, indicating the lower prevalence of drug-resistant HBV in this group than group A (Fig. 4B). The results were confirmed by PCR Sanger sequencing. In group C, 360 samples (90%, 360/400) were confirmed to be HBV positive, and all of them gave MMCA results. Two samples (0.56%, 2/360) were found to contain an LMV monoresistance mutation, indicating the rare occurrence of drug-resistant HBV in this group compared with its rate of occurrence in groups A and B (Fig. 4C). The results were again confirmed by PCR Sanger sequencing. Collectively, 114 of all 846 HBV-positive samples were found
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TABLE 1 Tm for each plasmid and ⌬Tm between wild-type plasmid and mutant plasmid Tm ⫾ 2SDs (°C) Resistance
Amino acid alteration
WTa (n ⫽ 10)
MTb (n ⫽ 10)
⌬⬎Tmc ⫾ 2SDs (°C) (n ⫽ 10)
LMV and LdT
rtL80I rtL80V
52.5 ⫾ 1.10 52.5 ⫾ 1.10
58.1 ⫾ 0.42 58.1 ⫾ 0.48
⫺5.65 ⫾ 1.06 ⫺5.70 ⫾ 1.26
LMV
rtV173G rtV173L
59.1 ⫾ 0.32 59.1 ⫾ 0.32
51.6 ⫾ 0.32 55.1 ⫾ 0.32
7.50 ⫾ 0.47 4.00 ⫾ 0.47
LMV and LdT
rtL180M
60.5 ⫾ 0.32
65.0 ⫾ 0.32
⫺4.60 ⫾ 0.42
ADV and LdT
rtA181T rtA181V
60.5 ⫾ 0.32 60.5 ⫾ 0.32
55.5 ⫾ 0.47 52.5 ⫾ 0.32
4.95 ⫾ 0.57 7.90 ⫾ 0.42
ETV
rtT184A rtT184S rtT184L rtT184I rtT184C rtT184F rtT184M rtT184G
58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42
54.0 ⫾ 0.32 54.6 ⫾ 0.63 49.0 ⫾ 0.32 47.5 ⫾ 0.32 46.5 ⫾ 0.63 46.5 ⫾ 0 46.5 ⫾ 0.32 45.5 ⫾ 0.32
4.65 ⫾ 0.48 4.00 ⫾ 0.81 9.65 ⫾ 0.72 11.15 ⫾ 0.48 12.00 ⫾ 0.48 12.10 ⫾ 0.42 12.05 ⫾ 0.56 13.05 ⫾ 0.32
TDF
rtA194T
68.2 ⫾ 0.48
64.1 ⫾ 0.42
4.05 ⫾ 0.74
ETV
rtS202C rtS202G rtS202I
60.0 ⫾ 0.47 60.0 ⫾ 0.47 60.0 ⫾ 0.47
53.6 ⫾ 0.32 53.1 ⫾ 0.42 54.1 ⫾ 0.42
6.45 ⫾ 0.56 6.90 ⫾ 0.63 5.90 ⫾ 0.63
LMV and LdT
rtM204I rtM204S rtM204V
63.6 ⫾ 0.32 63.6 ⫾ 0.32 63.6 ⫾ 0.32
53.5 ⫾ 0.32 49.6 ⫾ 0.42 56.0 ⫾ 0.32
10.10 ⫾ 0.42 13.95 ⫾ 0.56 7.60 ⫾ 0.42
TDF ADV
rtI233V rtN236T
53.9 ⫾ 0.42 52.2 ⫾ 0.67
58.3 ⫾ 0.84 56.2 ⫾ 0.67
⫺4.40 ⫾ 0.79 ⫺4.00 ⫾ 1.05
a
WT, wild type. MT, mutant type. c ⌬Tm, Tm for wild type ⫺ Tm for mutant type. b
to contain anti-HBV drug resistance mutations by the MMCA assay. The concordance with PCR Sanger sequencing was 99.3% (840/846), with a kappa value of 0.968 (95% confidence interval, 0.972 to 0.996). The concordance for the samples containing mutant and wild-type HBV DNA was 94.7% (108/114) and 100% (732/732), respectively.
Finally, we estimated the turnaround time and cost for the MMCA assay. One round of the assay, which can process 48 samples, required 10 min for template addition, 2 h and 10 min for PCR and melting analysis, and 10 min for mutation reading. Thus, the entire assay could be finished within 3 h when HBV DNA and the PCR master mix are ready for use. Two rounds of MMCA
FIG 2 Readout of TET channel in reaction A for serial dilutions of wild-type plasmids and rtM204V mutant plasmids. Red lines, melting curve for plasmids with 5 copies per reaction; black lines, melting curves for plasmids with concentrations of 5 ⫻ 106, 5 ⫻ 105, 5 ⫻ 104, 5 ⫻ 103, 5 ⫻ 102, and 5 ⫻ 101 copies per reaction; gray lines, negative control.
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FIG 3 Comparison of PCR Sanger sequencing, COLD-PCR Sanger sequencing, and the MMCA method for minority rtM204V mutation detection. (Left) The readouts for minor subpopulations of the rtM204V variant obtained using PCR Sanger sequencing (left), COLD-PCR Sanger sequencing (middle), and the MMCA assay (right) are shown. Green curves in right panels, melting curves for wild-type plasmid; black curves in right panels, melting curves for minority rtM204V mutant plasmid.
assay could easily be completed in one working day, during which 96 samples could be processed with a single real-time PCR instrument. The cost of materials for the MMCA assay was estimated to be $4 for each sample. The overall cost of materials for one sample would be less than $10 when other consumables, e.g., DNA extraction reagents and tips, are included. The cost of instrumentation for our assay depends on the thermocycler used. In this study, we used a Bio-Rad CFX-96 thermocycler, which costs approximately $30,000. In comparison, the cost of instrumentation for other HBV drug resistance assays currently available is much higher. For example, the instrument used to perform the line probe assay (Auto-LIA 48; Fujirebio Europe, Ghent, Belgium) is close to $100,000, and the cost of the genetic analyzer used for Sanger sequencing (Applied Biosystems 3500 genetic analyzer; Thermo
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Fisher Scientific, Waltham, MA, USA) is at least $150,000. Clearly, the short turnaround time and low financial barrier of the MMCA assay would facilitate its implementation in LMICs. DISCUSSION
We examined a two-reaction MMCA assay for anti-HBV drug resistance mutations. The MMCA assay could reliably detect 24 mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The assay had a limit of detection of 5 copies per reaction. The ability of MMCA to detect minor variant populations was better than that of PCR Sanger sequencing and equivalent to that of COLD-PCR Sanger sequencing. Clinical validation studies using 846 valid serum samples obtained from different patient populations demonstrated the accu-
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TABLE 2 Mutations detected by MMCA and PCR Sanger sequencing in 212 clinical samples from NA therapy population in China No. of mutations detected by: MMCA a
Agreement (%) Sequencing
b
Amino acid alteration
WT
MT
WT
MT
Mutation
Total
rtL80I/V rtV173L rtV173G rt(L/V)180M rtA181T rtA181V rtT184(A/C/F/G/I/L/M/S) rtA194T rtS202(C/G/I) rtM204I rtM204V rtM204S rtI233V rtN236T
192 211 211 172 206 203 208 212 210 158 185 212 212 197
20 1 1 40 6 9 4 0 2 54 27 0 0 15
194 211 211 172 206 203 208 212 211 158 185 212 212 200
18 1 1 40 6 9 4 0 1 54 27 0 0 12
90.0 100 100 100 100 100 100 100 50.0 100 100 100 100 80.0
99.1 100 100 100 100 100 100 100 99.5 100 100 100 100 98.6
a b
WT, wild type. MT, mutant type.
racy, suitability, and feasibility of the MMCA assay in clinical settings. The establishment and successful implementation of assays to detect anti-HBV drug resistance in resource-limited settings depend on a well-planned process of adaptation to and integration into relevant national strategies and guidelines (20). HBV DNA quantification is important for decisions on the initiation of antiviral therapy and monitoring of individuals during antiviral therapy (21). HBV DNA viral load assays based on real-time PCR have been increasingly available in LMICs (22). Thus, detection of antiHBV drug resistance mutations could be facilitated by utilization of the same platform. In this regard, the MMCA assay could be directly added to current settings for HBV viral load detection and is a preferred choice for CHB management over assays that need extra instruments. Moreover, the MMCA assay is a closed-tube procedure that requires no post-PCR manipulations, which not only lowers the risk of false-positive results and simplifies the operating procedure but also saves the costs for the facility and staff training. The reliability of the MMCA assay was well characterized by its analytical performance. First, the MMCA assay covers almost all
important anti-HBV drug resistance mutations. It covers 24 mutations at 10 amino acid positions, excluding 3 mutations [rtM250(I/V/L)] at residue rtM250 detected by the HBV DR v.3 assay (15). The rtM250(I/V/L) variation is a member of multiple mutations (T184, S202, or M250) that confer ETV resistance in an LVD-resistant HBV background with changes that have already occurred at L180M and M204(I/V) (23). However, rtM250(I/V/L) is rare compared with the incidence of mutations at T184 and S202 (23, 24), and its accurate detection is challenging due to the presence of various polymorphic sites in the neighboring nucleotides. For example, Degertekin et al. (15) reported that among 240 detected mutations, the HBV DR v.3 assay detected M250L in three samples, but sequencing analysis or follow-up studies could not confirm this result for any of them. Thus, omission of rtM250 would not compromise the overall performance of the MMCA assay. Second, the MMCA assay could detect minor variant subpopulations with a sensitivity that was equal to that of COLD-PCR Sanger sequencing but better than that of PCR Sanger sequencing. The plasmid DNA experiments showed that both MMCA and COLD-PCR Sanger sequencing could reliably detect rtM204V when it was present at a prevalence of as low as 5% of the total
FIG 4 Mutant HBV strains detected by MMCA from three different groups of patients. (A) Mutant HBV strains detected from NA therapy patient population in China; (B) mutant HBV strains detected from health examination population in China; (C) mutant HBV strains detected from health examination population in Pakistan. Only results for those HBV-positive samples confirmed by this study (total number, 846) are included.
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population, while conventional PCR Sanger sequencing could detect rtM204V only when it was present at a prevalence of greater than 10%. When it was applied to clinical samples, MMCA detected mutations in 6 more samples from the group A population than PCR Sanger sequencing, and all of them were confirmed by COLD-PCR Sanger sequencing. Although no systematic comparison with COLD-PCR Sanger sequencing was made in this work, our results demonstrate that MMCA can be used for the early detection of the emergence of anti-HBV drug resistance mutations. Third, MMCA had a high reproducibility for the determination of Tm values. Indeed, in these experiments, MMCA provided consistent Tm values regardless of the template amount (25). In this study, the SD values for Tm were smaller than 1°C, while the change in Tm (⌬Tm) values were larger than 4°C, ensuring the unambiguous detection of the mutations. Polymorphic nucleotides might occur in the proximity of the mutation and exert an influence on the Tm values. By using flexible design strategies in probe design, such an influence could be eliminated (26). Finally, the high analytical sensitivity of the MMCA assay ensured its applicability to samples with varied amounts of HBV DNA. The robustness of the MMCA assay was demonstrated by testing three groups of clinical samples. Of the total of 846 HBVpositive samples, 840 (99.3%) samples gave results concordant with those of PCR Sanger sequencing. Specifically, of the overall 8,460 amino acids analyzed, 8,454 (99.9%) amino acids gave results identical to those obtained by PCR Sanger sequencing. Furthermore, the 6 samples with inconsistent results were found to contain minor mutant subpopulations undetected by PCR Sanger sequencing, and their presence was confirmed by COLD-PCR Sanger sequencing. These results are in line with those of our previous studies that MMCA never missed a single mutation detected by PCR Sanger sequencing (18). The three groups of samples were representative of those from two types of patients, treated (group A) and untreated (groups B and C). Among the treated patients, 48.6% were found to be infected with isolates that contained drug resistance mutations. In contrast, among the untreated group, isolates from only 1.7% contained drug resistance mutations. This result was expected, as most anti-HBV drug resistance mutations are produced by long-term NA treatment. The frequency of mutation was as follows: LMV monoresistance ⬎ ADV monoresistance ⬎ LMV and ETV resistance ⬎ LMV and ADV resistance. This order is understandable, as LMV was the first NA drug cleared by FDA in 1998 (27), followed by ADV (28) and ETV (29). Also, LMV and ADV resistance can be caused by a single mutation, whereas ETV resistance is caused by multiple mutations. Notably, among the untreated patients, the Chinese groups had more mutation-containing samples than the Pakistani group (3.3% versus 0.56%), indicating that Chinese people have a greater chance of being infected by drug-resistant HBV than Pakistani people. This result reflects the fact that NAs are more commonly used to treat CHB in China than in Pakistan. In conclusion, the MMCA assay could reliably detect 24 antiHBV drug resistance mutations. It was more sensitive than PCR Sanger sequencing in the detection of minor variant subpopulations. Because the MMCA assay is technically simple, rapid, inexpensive, and amenable to high throughput, it could be recommended as a tool of choice for detection of anti-HBV resistance mutations in LMICs with high CHB burdens.
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ACKNOWLEDGMENTS We thank Xinlin Zhao for critical reading of the manuscript. The assay described has been transferred to Zeesan Biotech from Xiamen University. Q.L. holds equity interest in Zeesan Biotech. This work was supported in part by the National Natural Science Foundation (no. 81271929 to Q.L.) and the Key Project of Cooperation Program for University and Industry of Fujian Province (no. 2014Y4007 to Y.L.).
FUNDING INFORMATION This work, including the efforts of Yiqun Liao, was funded by Key Project of Cooperation Program for University and Industry of Fujian Province (2014Y4007). This work, including the efforts of Qingge Li, was funded by National Natural Science Foundation of China (NSFC) (81271929).
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Detection of Anti-Hepatitis B Virus Drug Resistance Mutations Based on Multicolor Melting Curve Analysis Yi Mou,a,b Muhammad Ammar Athar,a,b Yuzhen Wu,a,b Ye Xu,a,b Jianhua Wu,c Zhenxing Xu,c Zulfiqar Hayder,d Saeed Khan,e Muhammad Idrees,f Muhammad Israr Nasir,g Yiqun Liao,a,h Qingge Lia,b State Key Laboratory of Cellular Stress Biology, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, Engineering Research Center of Molecular Diagnostics of the Ministry of Education, School of Life Sciences, Xiamen University, Xiamen, Fujian, Chinaa; Shenzhen Research Institute of Xiamen University, Shenzhen, Guangdong, Chinab; Xiamen Hospital of Traditional Chinese Medicine, Xiamen, Fujian, Chinac; Department of Pathology, Quid-e-Azam Medical College, Bahawalpur, Punjab, Pakistand; Department of Molecular Pathology, Dow University of Health Sciences, Karachi, Pakistane; Center for Applied Molecular Biology, University of the Punjab, Lahore, Pakistanf; Department of Molecular Pathology, Liaquat National Hospital, Karachi, Pakistang; School of Public Health, Xiamen University, Xiamen, Fujian, Chinah
Detection of anti-hepatitis B virus (HBV) drug resistance mutations is critical for therapeutic decisions for chronic hepatitis B virus infection. We describe a real-time PCR-based assay using multicolor melting curve analysis (MMCA) that could accurately detect 24 HBV nucleotide mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The two-reaction assay had a limit of detection of 5 copies per reaction and could detect a minor mutant population (5% of the total population) with the reverse transcriptase M204V amino acid mutation in the presence of the major wild-type population when the overall concentration was 104 copies/l. The assay could be finished within 3 h, and the cost of materials for each sample was less than $10. Clinical validation studies using three groups of samples from both nucleos(t)ide analog-treated and -untreated patients showed that the results for 99.3% (840/846) of the samples and 99.9% (8,454/8,460) of the amino acids were concordant with those of Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (PCR Sanger sequencing). HBV DNA in six samples with mixed infections consisting of minor mutant subpopulations was undetected by the PCR Sanger sequencing method but was detected by MMCA, and the results were confirmed by coamplification at a lower denaturation temperature-PCR Sanger sequencing. Among the treated patients, 48.6% (103/212) harbored viruses that displayed lamivudine monoresistance, adefovir monoresistance, entecavir resistance, or lamivudine and adefovir resistance. Among the untreated patients, the Chinese group had more mutation-containing samples than did the Pakistani group (3.3% versus 0.56%). Because of its accuracy, rapidness, wide-range coverage, and cost-effectiveness, the real-time PCR assay could be a robust tool for the detection if anti-HBV drug resistance mutations in resource-limited countries.
H
epatitis B is caused by the hepatitis B virus (HBV), an enveloped DNA virus that infects the liver, causing hepatocellular necrosis and inflammation (1). Chronic hepatitis B (CHB) infection affects approximately 248 million people worldwide and is a leading cause of liver-related morbidity and mortality, particularly in low- and middle-income countries (LMICs) (2). Patients with CHB can be successfully treated using nucleos(t)ide analogs (NAs), but drug-resistant HBV mutants frequently arise, leading to treatment failure and progression to liver disease (3). The development of drug resistance begins with mutations in the HBV polymerase gene, followed by an increase in the viral load and serum alanine aminotransferase levels several weeks to months later (4). Detection of drug resistance mutations is thus critical in prompt decision making for new therapeutic regimes (5, 6). A method enabling detection of NA resistance mutations should be reliable, rapid, and, in particular, easy to use and cost-effective when the aim is for it to be used in high-CHB-burden countries, which are often undeveloped and resource limited. Many methods for the detection of anti-HBV drug resistance mutations have been developed. However, these methods are almost exclusively performed by highly skilled technicians in wellequipped referral hospitals. This limitation restricts the use of anti-HBV drug resistance assays to only a small number of patients, which offers little help for CHB management program in LMICs. Sanger sequencing of the PCR amplicon from the HBV reverse transcriptase region (referred to here as PCR Sanger sequencing)
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is currently the “gold standard” method for the detection of antiHBV drug resistance mutations. It is advantageous for the accurate detection of all nucleotide mutations, including novel mutations which have never been reported before. However, the Sanger sequencer is often unaffordable for the local hospitals that admit a majority of CHB patients in LMICs. Moreover, the sequencing procedure is complex and lengthy and requires very careful operations to avoid contamination from PCR amplicons. This is also true for other sequencing-based assays, such as pyrosequencing (7), coamplification at a lower denaturation temperature (COLD)-
Received 27 February 2016 Returned for modification 12 May 2016 Accepted 5 August 2016 Accepted manuscript posted online 17 August 2016 Citation Mou Y, Athar MA, Wu Y, Xu Y, Wu J, Xu Z, Hayder Z, Khan S, Idrees M, Nasir MI, Liao Y, Li Q. 2016. Detection of anti-hepatitis B virus drug resistance mutations based on multicolor melting curve analysis. J Clin Microbiol 54:2661–2668. doi:10.1128/JCM.00439-16. Editor: Y.-W. Tang, Memorial Sloan-Kettering Cancer Center Address correspondence to Yiqun Liao, [email protected], or Qingge Li, [email protected]. Y.M. and M.A.A. contributed equally to this article. Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.00439-16. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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PCR Sanger sequencing (8–10), and next-generation sequencing (11, 12), as well as the high-end instrument-based assays, such as DNA microchip (13) and mass spectrometry (14) assays. Several simpler and more cost-effective assays for detecting anti-HBV drug resistance mutations have been developed. For example, the line probe assay (HBV DR v.3; Fujirebio Europe, Ghent, Belgium) is a reverse line blot assay that detects 27 mutations at 11 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The HBV DR v.3 assay is technically simple, rapid, and amenable to high throughput (15). However, as an open-tube assay, it requires multiple post-PCR hybridization and visualization steps, which are prone to amplicon contamination, which leads to the generation of false-positive results. Real-time PCR is an excellent platform of choice for clinical diagnosis because of its closed-tube detection format, which makes it easy to use and rapid and which results in decreased amplicon contamination. A realtime amplification refractory mutation system (ARMS) PCR for the detection of six HBV resistance mutations has been developed. Nevertheless, it is far from practical due to the limited coverage of mutation types (16). We previously reported a multiplex real-time PCR detection strategy based on multicolor melting curve analysis (MMCA) using dually labeled, self-quenched probes (17). Up to 16 genetic mutations could be detected in a single reaction by this strategy (18). In the present study, we describe a two-reaction MMCA assay that can detect 24 anti-HBV drug resistance mutations at 10 amino acid positions. We systematically evaluated its analytical performance, including its mutation detection accuracy, its analytical sensitivity, and, in particular, its ability to detect minor variants. We further evaluated its clinical performance by analyzing samples from a group of 212 CHB patients who had received nucleos(t)ide analog treatment, samples from a second group of 276 HBV-positive people who were selected from among those receiving a routine health examination, and a third group of 400 HBV-positive samples collected from individuals in Pakistan. The results were compared with those of PCR Sanger sequencing and confirmed by COLD-PCR Sanger sequencing. MATERIALS AND METHODS Clinical samples. In total, 888 serum samples were collected from the same number of deidentified patients. These samples were labeled HBV positive when they were received. The clinical samples were divided into three groups. Samples in group A (212 samples) were collected from patients who had received or were undergoing treatment with NAs at the Xiamen Hospital of Traditional Chinese Medicine (Xiamen, China). Samples in group B (276 samples) were collected in the same hospital from individuals who were being seen for a routine health examination and had never received NA drugs. Samples in group C (400 samples) were collected from four medical colleges or hospitals in Pakistan, including Quid-e-Azam Medical College, Dow University of Health Sciences, University of the Punjab, and Liaquat National Hospital. An exemption from full committee review for human subject studies was granted by the Xiamen University Research Ethics Committee since only coded samples collected for other procedures were used in the present work. HBV DNA was extracted from the serum samples using a Lab-Aid 820 virus DNA isolation kit (Zeesan Biotech Ltd., Xiamen, China). The DNA extracts were used for both MMCA and PCR Sanger sequencing. Before mutation detection, the presence of HBV in the sample was confirmed by use of an HBV real-time PCR quantification kit (Zhijiang Biotech Ltd., Shanghai, China). Plasmid DNA. Wild-type plasmid DNA was prepared by use of an HBV-positive sample obtained from the Xiamen Hospital of Tradi-
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tional Chinese Medicine. An amplicon of 828 bp from reverse transcriptase amino acid 14 (rt14) to rt289 was generated using forward primer 5=-ATCAGGACTCCTAGGACC-3= and reverse primer 5=-TCGTTGACA TACTTTCCAATC-3=. Sequencing analysis confirmed that the amplicon corresponded to that from the HBV isolate with GenBank accession number GQ377639.1 (http://www.ncbi.nlm.nih.gov/) with one non-drug resistance mutation (A ¡ G) on the third base of rt255. A plasmid carrying the wild-type HBV sequence was constructed by cloning the amplicon into the pMD18-T vector using a TA cloning kit (TaKaRa, Dalian, China). Plasmids carrying mutant DNA sequences were constructed from the wild-type plasmid by site-directed mutagenesis (19). In total, 24 plasmids with 24 mutations, i.e., rtL80I, rtL80V, rtV173L, rtV173G, rt(L/V)180M, rtA181T, rtA181V, rtT184A, rtT184C, rtT184F, rtT184G, rtT184I, rtT184L, rtT184M, rtT184S, rtA194T, rtS202C, rtS202G, rtS202I, rtM204I, rtM204V, rtM204S, rtI233V, and rtN236T, were prepared. The drug resistance nucleotide mutation(s) corresponding to each amino acid alteration was determined (see Table S1 in the supplemental material). The plasmid DNA in 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA (TE buffer) was quantified using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Montchanin, DE). MMCA assay. The MMCA assay has two reactions that detect 24 mutations in HBV that confer resistance against the five FDA-approved antiHBV NA drugs, including lamivudine (LMV), adefovir (ADV), entecavir (ETV), tenofovir (TDF), and telbivudine (LdT). Reaction A detects 10 mutations, including rtL80I, rtL80V, rt(L/V)180M, rtA181T, rtA181V, rtA194T, rtM204I, rtM204V, rtM204S, and rtN236T. Reaction B detects 14 mutations, including rtV173L, rtV173G, rtT184A, rtT184C, rtT184F, rtT184G, rtT184I, rtT184L, rtT184M, rtT184S, rtS202C, rtS202G, rtS202I, and rtI233V. Each MMCA assay reaction mixture contains 2.5 l of 10⫻ Taq HS buffer (TaKaRa), 0.2 l of Taq HS (5 units/l), 0.2 l of deoxynucleoside triphosphates (dNTPs; 25 mM), 0.1 l of primer-probe mix, 17 l of deionized H2O, and 5 l of the HBV DNA template. PCR and melting curve analysis were performed in a Bio-Rad CFX-96 thermocycler (BioRad, Hercules, CA). PCR was started with denaturation at 95°C for 5 min and was followed by 45 cycles of 95°C for 15 s, 52°C for 15 s, and 72°C for 20 s. Melting curve analysis began with denaturation at 95°C for 60 s and hybridization at 30°C for 60 s, followed by a gradual temperature increase from 30°C to 85°C in increments of 0.5°C/s. The fluorescence from the 6-carboxyfluorescein (FAM; absorbance [abs] and emission [em], 495 nm and 520 nm, respectively), hexachloro-6-carboxyfluorescein (HEX; abs and em, 521 nm and 536 nm, respectively), 6-carboxy-Xrhodamine (ROX; abs and em, 586 nm and 610 nm, respectively), and indodicarbocyanine 5 (Cy5; abs and em, 647 nm and 667 nm, respectively) channels was recorded. PCR and COLD-PCR for Sanger sequencing. Each 25-l PCR mixture was prepared as described above and contained 2.5 l of 10⫻ Taq HS buffer, 0.2 l of Taq HS, 0.2 l of dNTPs, 0.1 l of primer mix, 17 l of deionized H2O, and 5 l of HBV DNA. The primer mix was composed of 20 pM each forward primer 5=-ATCAGGACTCCTAGGACC-3= and reverse primer 5=-TCGTTGACATACTTTCCAATC-3=. Conventional PCR was carried out in a T3 thermocycler (Biometra, Göttingen, Germany) using a program of 95°C for 5 min, followed by 45 cycles of 95°C for 15 s, 52°C for 15 s, and 72°C for 40 s. COLD-PCR was performed essentially as previously described (10). Briefly, 5 l of the HBV DNA template was added to a 20-l PCR mixture containing 2.5 l of 10⫻ Taq HS buffer, 0.2 l of Taq HS (5 unit/l), 0.1 l of primer mix (50 M for each), 0.2 l of dNTPs (25 mM), and 17 l of deionized H2O. Amplification was carried out in a RotorGene Q realtime PCR system (Qiagen, Hilden, Germany) under the following conditions: 95°C for 10 min; 10 cycles of 95°C for 15 s, 50°C for 30 s, and 72°C for 1 min; 72°C for 7 min; 95°C for 2 min; and finally, 30 cycles of 95°C for 15 s, 70°C for 1 min, 77°C for 5 s, 50°C for 30 s, and 72°C for 1 min. The amplified products were sent out for bidirectional Sanger sequencing (Sangon Inc., Shanghai, China).
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Real-Time PCR Detection of Drug-Resistant HBV
FIG 1 Readout for a wild-type plasmid and 24 mutant plasmids using the MMCA method. (Top) Typical readout for reaction A; (bottom) typical readout for reaction B. Black lines, melting curve of wild-type plasmid; gray lines, melting curves of 24 mutant plasmids. WT, wild type; ⫺dF/dt, negative derivative of fluorescence over temperature; TET, tetrachloro-6-carboxyfluorescein.
RESULTS
We evaluated the analytical performance of the MMCA assay by using a plasmid carrying wild-type HBV DNA (wild-type plasmid) and 24 plasmids carrying mutant HBV DNA (mutant plasmids) as mimics of the respective wild-type and mutant HBV DNA samples. For each plasmid, we prepared serial 10-fold dilutions in TE ranging from 106 to 100 copies/l. The MMCA assay showed that for each plasmid, a unique melting temperature (Tm) value could be obtained regardless of the concentration. Figure 1 shows the melting curves of all the plasmids at 10 copies/l. Ten repeated detections at this concentration provided the average Tm and standard deviation (SD) for each plasmid (Table 1). All the Tm values for the mutant plasmids had a ⬎4°C difference from the Tm value for the wild-type plasmid, and the SD values were less than 1°C. As the Tm resolution of the MMCA assay was 0.5°C, all mutant plasmids investigated could be unequivocally differentiated from the wild-type plasmid. The analytical sensitivity of the assay was obtained through 10 repeated detections of each plasmid at 10 copies/l and 1 copy/l. The results showed that all plasmids at 1 copy/l or 5 copies per reaction could be detected (Fig. 2), and the limit of detection of the MMCA assay was thus 1 copy/l, or 5 copies per reaction. To test the ability of MMCA assay to detect minor mutant subpopulations, we prepared a series of mixtures containing plasmids carrying the wild type and the rtM204V mutant. The overall template concentration was set to 104 copies/l, and the percentage of the population that was the rtM204V mutant was 0%, 5%, 10%, and 20%. These mixtures were subjected to MMCA, PCR Sanger sequencing, and COLD-PCR Sanger sequencing in parallel. The results showed that both the MMCA assay and COLDPCR Sanger sequencing could detect the rtM204V mutant subpopulation when it was present at proportions of 5% and above, whereas PCR Sanger sequencing method could detect the subpopulation when it was present at proportions of 10% and above
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(Fig. 3), demonstrating that MMCA has a sensitivity for the detection of minor mutant subpopulations equal to that of COLD-PCR Sanger sequencing, which is more sensitive than PCR Sanger sequencing. To evaluate the clinical performance of MMCA, we used three different groups of patient samples. The results showed that all samples in group A were confirmed to be HBV positive. The mutations detected are listed in Table 2. Of the 212 samples, 103 (48.6%) were found to contain anti-HBV drug resistance mutations, indicating the widespread prevalence of drug resistance in this group. An LMV monoresistance mutation (70.9%, 73/103) was the most frequently detected mutation, followed by an ADV monoresistance mutation (21.4%, 22/103), an LMV and ETV resistance mutation (4.9%, 5/103), and an LMV and ADV resistance mutation (2.9%, 3/103) (Fig. 4A). MMCA detected 6 more samples with mutations than PCR Sanger sequencing did, and these were found to carry minor mutant subpopulations that escaped detection by PCR Sanger sequencing. These results were confirmed by COLD-PCR Sanger sequencing. In group B, 274 samples (99.3%, 274/276) were confirmed to be HBV positive, and all of these gave MMCA results. Nine samples (3.3%, 9/274) were found to contain anti-HBV drug resistance mutations, including 7 LMV monoresistance mutations and 2 ADV monoresistance mutations, indicating the lower prevalence of drug-resistant HBV in this group than group A (Fig. 4B). The results were confirmed by PCR Sanger sequencing. In group C, 360 samples (90%, 360/400) were confirmed to be HBV positive, and all of them gave MMCA results. Two samples (0.56%, 2/360) were found to contain an LMV monoresistance mutation, indicating the rare occurrence of drug-resistant HBV in this group compared with its rate of occurrence in groups A and B (Fig. 4C). The results were again confirmed by PCR Sanger sequencing. Collectively, 114 of all 846 HBV-positive samples were found
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TABLE 1 Tm for each plasmid and ⌬Tm between wild-type plasmid and mutant plasmid Tm ⫾ 2SDs (°C) Resistance
Amino acid alteration
WTa (n ⫽ 10)
MTb (n ⫽ 10)
⌬⬎Tmc ⫾ 2SDs (°C) (n ⫽ 10)
LMV and LdT
rtL80I rtL80V
52.5 ⫾ 1.10 52.5 ⫾ 1.10
58.1 ⫾ 0.42 58.1 ⫾ 0.48
⫺5.65 ⫾ 1.06 ⫺5.70 ⫾ 1.26
LMV
rtV173G rtV173L
59.1 ⫾ 0.32 59.1 ⫾ 0.32
51.6 ⫾ 0.32 55.1 ⫾ 0.32
7.50 ⫾ 0.47 4.00 ⫾ 0.47
LMV and LdT
rtL180M
60.5 ⫾ 0.32
65.0 ⫾ 0.32
⫺4.60 ⫾ 0.42
ADV and LdT
rtA181T rtA181V
60.5 ⫾ 0.32 60.5 ⫾ 0.32
55.5 ⫾ 0.47 52.5 ⫾ 0.32
4.95 ⫾ 0.57 7.90 ⫾ 0.42
ETV
rtT184A rtT184S rtT184L rtT184I rtT184C rtT184F rtT184M rtT184G
58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42 58.6 ⫾ 0.42
54.0 ⫾ 0.32 54.6 ⫾ 0.63 49.0 ⫾ 0.32 47.5 ⫾ 0.32 46.5 ⫾ 0.63 46.5 ⫾ 0 46.5 ⫾ 0.32 45.5 ⫾ 0.32
4.65 ⫾ 0.48 4.00 ⫾ 0.81 9.65 ⫾ 0.72 11.15 ⫾ 0.48 12.00 ⫾ 0.48 12.10 ⫾ 0.42 12.05 ⫾ 0.56 13.05 ⫾ 0.32
TDF
rtA194T
68.2 ⫾ 0.48
64.1 ⫾ 0.42
4.05 ⫾ 0.74
ETV
rtS202C rtS202G rtS202I
60.0 ⫾ 0.47 60.0 ⫾ 0.47 60.0 ⫾ 0.47
53.6 ⫾ 0.32 53.1 ⫾ 0.42 54.1 ⫾ 0.42
6.45 ⫾ 0.56 6.90 ⫾ 0.63 5.90 ⫾ 0.63
LMV and LdT
rtM204I rtM204S rtM204V
63.6 ⫾ 0.32 63.6 ⫾ 0.32 63.6 ⫾ 0.32
53.5 ⫾ 0.32 49.6 ⫾ 0.42 56.0 ⫾ 0.32
10.10 ⫾ 0.42 13.95 ⫾ 0.56 7.60 ⫾ 0.42
TDF ADV
rtI233V rtN236T
53.9 ⫾ 0.42 52.2 ⫾ 0.67
58.3 ⫾ 0.84 56.2 ⫾ 0.67
⫺4.40 ⫾ 0.79 ⫺4.00 ⫾ 1.05
a
WT, wild type. MT, mutant type. c ⌬Tm, Tm for wild type ⫺ Tm for mutant type. b
to contain anti-HBV drug resistance mutations by the MMCA assay. The concordance with PCR Sanger sequencing was 99.3% (840/846), with a kappa value of 0.968 (95% confidence interval, 0.972 to 0.996). The concordance for the samples containing mutant and wild-type HBV DNA was 94.7% (108/114) and 100% (732/732), respectively.
Finally, we estimated the turnaround time and cost for the MMCA assay. One round of the assay, which can process 48 samples, required 10 min for template addition, 2 h and 10 min for PCR and melting analysis, and 10 min for mutation reading. Thus, the entire assay could be finished within 3 h when HBV DNA and the PCR master mix are ready for use. Two rounds of MMCA
FIG 2 Readout of TET channel in reaction A for serial dilutions of wild-type plasmids and rtM204V mutant plasmids. Red lines, melting curve for plasmids with 5 copies per reaction; black lines, melting curves for plasmids with concentrations of 5 ⫻ 106, 5 ⫻ 105, 5 ⫻ 104, 5 ⫻ 103, 5 ⫻ 102, and 5 ⫻ 101 copies per reaction; gray lines, negative control.
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FIG 3 Comparison of PCR Sanger sequencing, COLD-PCR Sanger sequencing, and the MMCA method for minority rtM204V mutation detection. (Left) The readouts for minor subpopulations of the rtM204V variant obtained using PCR Sanger sequencing (left), COLD-PCR Sanger sequencing (middle), and the MMCA assay (right) are shown. Green curves in right panels, melting curves for wild-type plasmid; black curves in right panels, melting curves for minority rtM204V mutant plasmid.
assay could easily be completed in one working day, during which 96 samples could be processed with a single real-time PCR instrument. The cost of materials for the MMCA assay was estimated to be $4 for each sample. The overall cost of materials for one sample would be less than $10 when other consumables, e.g., DNA extraction reagents and tips, are included. The cost of instrumentation for our assay depends on the thermocycler used. In this study, we used a Bio-Rad CFX-96 thermocycler, which costs approximately $30,000. In comparison, the cost of instrumentation for other HBV drug resistance assays currently available is much higher. For example, the instrument used to perform the line probe assay (Auto-LIA 48; Fujirebio Europe, Ghent, Belgium) is close to $100,000, and the cost of the genetic analyzer used for Sanger sequencing (Applied Biosystems 3500 genetic analyzer; Thermo
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Fisher Scientific, Waltham, MA, USA) is at least $150,000. Clearly, the short turnaround time and low financial barrier of the MMCA assay would facilitate its implementation in LMICs. DISCUSSION
We examined a two-reaction MMCA assay for anti-HBV drug resistance mutations. The MMCA assay could reliably detect 24 mutations at 10 amino acid positions in the reverse transcriptase region of the HBV polymerase gene. The assay had a limit of detection of 5 copies per reaction. The ability of MMCA to detect minor variant populations was better than that of PCR Sanger sequencing and equivalent to that of COLD-PCR Sanger sequencing. Clinical validation studies using 846 valid serum samples obtained from different patient populations demonstrated the accu-
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TABLE 2 Mutations detected by MMCA and PCR Sanger sequencing in 212 clinical samples from NA therapy population in China No. of mutations detected by: MMCA a
Agreement (%) Sequencing
b
Amino acid alteration
WT
MT
WT
MT
Mutation
Total
rtL80I/V rtV173L rtV173G rt(L/V)180M rtA181T rtA181V rtT184(A/C/F/G/I/L/M/S) rtA194T rtS202(C/G/I) rtM204I rtM204V rtM204S rtI233V rtN236T
192 211 211 172 206 203 208 212 210 158 185 212 212 197
20 1 1 40 6 9 4 0 2 54 27 0 0 15
194 211 211 172 206 203 208 212 211 158 185 212 212 200
18 1 1 40 6 9 4 0 1 54 27 0 0 12
90.0 100 100 100 100 100 100 100 50.0 100 100 100 100 80.0
99.1 100 100 100 100 100 100 100 99.5 100 100 100 100 98.6
a b
WT, wild type. MT, mutant type.
racy, suitability, and feasibility of the MMCA assay in clinical settings. The establishment and successful implementation of assays to detect anti-HBV drug resistance in resource-limited settings depend on a well-planned process of adaptation to and integration into relevant national strategies and guidelines (20). HBV DNA quantification is important for decisions on the initiation of antiviral therapy and monitoring of individuals during antiviral therapy (21). HBV DNA viral load assays based on real-time PCR have been increasingly available in LMICs (22). Thus, detection of antiHBV drug resistance mutations could be facilitated by utilization of the same platform. In this regard, the MMCA assay could be directly added to current settings for HBV viral load detection and is a preferred choice for CHB management over assays that need extra instruments. Moreover, the MMCA assay is a closed-tube procedure that requires no post-PCR manipulations, which not only lowers the risk of false-positive results and simplifies the operating procedure but also saves the costs for the facility and staff training. The reliability of the MMCA assay was well characterized by its analytical performance. First, the MMCA assay covers almost all
important anti-HBV drug resistance mutations. It covers 24 mutations at 10 amino acid positions, excluding 3 mutations [rtM250(I/V/L)] at residue rtM250 detected by the HBV DR v.3 assay (15). The rtM250(I/V/L) variation is a member of multiple mutations (T184, S202, or M250) that confer ETV resistance in an LVD-resistant HBV background with changes that have already occurred at L180M and M204(I/V) (23). However, rtM250(I/V/L) is rare compared with the incidence of mutations at T184 and S202 (23, 24), and its accurate detection is challenging due to the presence of various polymorphic sites in the neighboring nucleotides. For example, Degertekin et al. (15) reported that among 240 detected mutations, the HBV DR v.3 assay detected M250L in three samples, but sequencing analysis or follow-up studies could not confirm this result for any of them. Thus, omission of rtM250 would not compromise the overall performance of the MMCA assay. Second, the MMCA assay could detect minor variant subpopulations with a sensitivity that was equal to that of COLD-PCR Sanger sequencing but better than that of PCR Sanger sequencing. The plasmid DNA experiments showed that both MMCA and COLD-PCR Sanger sequencing could reliably detect rtM204V when it was present at a prevalence of as low as 5% of the total
FIG 4 Mutant HBV strains detected by MMCA from three different groups of patients. (A) Mutant HBV strains detected from NA therapy patient population in China; (B) mutant HBV strains detected from health examination population in China; (C) mutant HBV strains detected from health examination population in Pakistan. Only results for those HBV-positive samples confirmed by this study (total number, 846) are included.
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population, while conventional PCR Sanger sequencing could detect rtM204V only when it was present at a prevalence of greater than 10%. When it was applied to clinical samples, MMCA detected mutations in 6 more samples from the group A population than PCR Sanger sequencing, and all of them were confirmed by COLD-PCR Sanger sequencing. Although no systematic comparison with COLD-PCR Sanger sequencing was made in this work, our results demonstrate that MMCA can be used for the early detection of the emergence of anti-HBV drug resistance mutations. Third, MMCA had a high reproducibility for the determination of Tm values. Indeed, in these experiments, MMCA provided consistent Tm values regardless of the template amount (25). In this study, the SD values for Tm were smaller than 1°C, while the change in Tm (⌬Tm) values were larger than 4°C, ensuring the unambiguous detection of the mutations. Polymorphic nucleotides might occur in the proximity of the mutation and exert an influence on the Tm values. By using flexible design strategies in probe design, such an influence could be eliminated (26). Finally, the high analytical sensitivity of the MMCA assay ensured its applicability to samples with varied amounts of HBV DNA. The robustness of the MMCA assay was demonstrated by testing three groups of clinical samples. Of the total of 846 HBVpositive samples, 840 (99.3%) samples gave results concordant with those of PCR Sanger sequencing. Specifically, of the overall 8,460 amino acids analyzed, 8,454 (99.9%) amino acids gave results identical to those obtained by PCR Sanger sequencing. Furthermore, the 6 samples with inconsistent results were found to contain minor mutant subpopulations undetected by PCR Sanger sequencing, and their presence was confirmed by COLD-PCR Sanger sequencing. These results are in line with those of our previous studies that MMCA never missed a single mutation detected by PCR Sanger sequencing (18). The three groups of samples were representative of those from two types of patients, treated (group A) and untreated (groups B and C). Among the treated patients, 48.6% were found to be infected with isolates that contained drug resistance mutations. In contrast, among the untreated group, isolates from only 1.7% contained drug resistance mutations. This result was expected, as most anti-HBV drug resistance mutations are produced by long-term NA treatment. The frequency of mutation was as follows: LMV monoresistance ⬎ ADV monoresistance ⬎ LMV and ETV resistance ⬎ LMV and ADV resistance. This order is understandable, as LMV was the first NA drug cleared by FDA in 1998 (27), followed by ADV (28) and ETV (29). Also, LMV and ADV resistance can be caused by a single mutation, whereas ETV resistance is caused by multiple mutations. Notably, among the untreated patients, the Chinese groups had more mutation-containing samples than the Pakistani group (3.3% versus 0.56%), indicating that Chinese people have a greater chance of being infected by drug-resistant HBV than Pakistani people. This result reflects the fact that NAs are more commonly used to treat CHB in China than in Pakistan. In conclusion, the MMCA assay could reliably detect 24 antiHBV drug resistance mutations. It was more sensitive than PCR Sanger sequencing in the detection of minor variant subpopulations. Because the MMCA assay is technically simple, rapid, inexpensive, and amenable to high throughput, it could be recommended as a tool of choice for detection of anti-HBV resistance mutations in LMICs with high CHB burdens.
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ACKNOWLEDGMENTS We thank Xinlin Zhao for critical reading of the manuscript. The assay described has been transferred to Zeesan Biotech from Xiamen University. Q.L. holds equity interest in Zeesan Biotech. This work was supported in part by the National Natural Science Foundation (no. 81271929 to Q.L.) and the Key Project of Cooperation Program for University and Industry of Fujian Province (no. 2014Y4007 to Y.L.).
FUNDING INFORMATION This work, including the efforts of Yiqun Liao, was funded by Key Project of Cooperation Program for University and Industry of Fujian Province (2014Y4007). This work, including the efforts of Qingge Li, was funded by National Natural Science Foundation of China (NSFC) (81271929).
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