Hepatitis C Virus Genotype Analyses in Chronic Hepatitis C Patients and Individuals With Spontaneous Virus Clearance Using a Newly Developed Serotyping Assay

Abstract

Background

We developed a novel HCV serotyping assay and detected the genotypes in chronic hepatitis C (CHC) patients and individuals with spontaneous viral clearance (SVC).

Methods

Nine hundred and ninety‐seven patients were enrolled in a previous study; their samples were genotyped originally using the molecular assays. Among them, 190 patients achieved sustained virological response; the post‐treatment samples were also serotyped. Moreover, 326 samples from follow‐up cohorts were serotyped, among whom 66 were from SVC individuals, and 260 from CHC patients.

Results

Nine hundred and fifty‐eight out of 997 samples were available for serotyping, among which 29 samples generated indeterminate serotyping results. The consistency between the genotyping and serotyping assays was 91.50% (850/929). The specificity and sensitivity were 98.45% and 88.77% for genotype 1, 96.42% and 93.97% for genotype 2, and 94.15% and 80.52% for non‐genotype 1 or 2. However, only 41 of 60 genotype‐6 samples were correctly serotyped. Little difference was found in the 190 paired serotyping results. No difference existed in the genotype distribution between the SVC and CHC groups (P = 0.08).

Conclusions

The assay provides an accurate alternative for determining HCV genotypes, whereas it is not recommended for detecting genotype 6. Furthermore, it facilitates identifying the genotypes in SVC individuals. HCV genotype has little impact on SVC.

Introduction

Hepatitis C virus (HCV) infection threatens the health of approximately 170 million people worldwide. Chronic HCV infection is a major cause of liver cirrhosis and hepatocellular carcinoma 1. Till date, HCV has been classified into seven genotypes and 67 subtypes 2. Genotype 1 was the most common (46%) globally, followed by genotype 3 (22%) and genotype 2 (13%). Genotypes 4, 5, and 6 are regionally distributed 3. HCV genotype is one of the crucial baseline factors to guide the treatment regimen and predict the outcome of the chronic hepatitis C (CHC) patients treated with peginterferon and ribavirin 4. With the rapid development of the direct antiviral agents (DAAs), however, the clinical importance of the HCV genotype has to be downgraded, as the activity of some potent DAAs or their combinations appears to be pan‐genotypic 5, 6. However, determination of the HCV genotype is of important clinical importance. First, the DAA treatment regimen is still genotype dependent. Current antiviral treatment regimens are based on different genotypes in the practice guidelines 7, 8, 9. For example, treatment‐naïve patients with genotype 3 should be treated with Sofosbuvir plus ribavirin for 24 weeks, while those with genotype 1 or 2 need only 12 week or even shorter treatment duration 10. It is more difficult to achieve sustained virological response (SVR) for treatment‐experienced patients with genotype 3, and optimal treatment is still under investigation 5. Second, a specific HCV‐resistance profile is related with different DAAs and varies depending on the genotypes 11. Third, the majority of the CHC patients, especially those from the Asian‐pacific regions, have not had access to the DAAs due to the high cost, whereas the efficacy of peginterferon plus ribavirin is satisfactory for them 9. Moreover, the distribution of viral genotypes reflects patterns of HCV transmission and human migration 12, 13. Therefore, determination of the HCV genotype is of clinical and epidemiological importance 14.

PCR based molecular methods such as direct sequencing, real‐time PCR and line probe hybridization assays have been used for HCV genotyping 14. But these methods are labor‐intensive, expensive and dependent of the thermal cycler, DNA hybridization or sequencing instrument. They must be performed at separated working areas to prevent potential aerosol contamination. Recently we have developed an HCV serotyping assay. It is based on enzyme‐linked immunosorbent assay (ELISA) technology and can identify genotypes 1, 2, 1/2 mixture, and non‐1/2 genotypes. It is simple, cheap and provides results rapidly. Furthermore, some individuals spontaneously clear the HCV after acute infection. They have undetectable serum HCV RNA, but the anti‐HCV antibodies are persistent for many years 15. The association between the viral genotype and spontaneous viral clearance (SVC) remains controversial 16, 17, 18, 19, 20. This assay provides a tool to determine the genotype in the SVC individuals.

In this study, we evaluated the performance of the novel serotyping assay in a large CHC population, and we also detected the genotypes in people with SVC to investigate the impact of the genotype on the spontaneous viral clearance.

Material and Methods

HCV Serotyping Assay and the Quality Controls

The serotyping assay was designed to be used to detect the serum or plasma samples containing anti‐HCV antibodies. It was based on a competitive inhibition ELISA principle. The core and NS4 antigens acted as the target genotype‐specific proteins, and the epitopes were predicted using the bioinformatics software Biosun 3.0 (Computational Biology Center, Beijing, China). Thereafter, the recombinant antigen fragments specific to genotypes 1 and 2 were expressed in the E. Coli HB101, purified, and then coated onto the 96‐well plate. Their amino acid sequences are listed in Table 1. Solutions containing competitive antigens against the antibodies of genotype 1, 2, and genotypes 1/2 were prepared as well. At the beginning of the assay, 10 μl of the competitive antigen was added to the well to block the corresponding genotype‐specific antibodies. Both competitive antigens were added to the completely neutralized well, and nothing was added to the non‐neutralized well. Thereafter, 80 μl of the dilution buffer and 10 μl of the serum samples were added to each well. The reaction was incubated for 30 min, during which the anti‐HCV antibodies in the sample were bound with the unblocked genotype‐specific coated antigens. After washing, the goat anti‐human IgG coupled with horseradish peroxidase (HRP) was added. Finally, the substrate was added after washing. The optical density (OD) value was measured at 450/630 nm of wavelength using the Sunrise microplate reader (Tecan, Grödig, Austria).

Table 1.

Recombinant coating antigens and competitive antigens for the serotyping assay

Antigen location	Positiona	Function	Amino acid sequence
Core	67–81	Genotype 1 specific, coated antigen	KARRPEGRTWAQPGY
Core	67–81	Genotype 2 specific, coated antigen	KDRRTTGKSWGRPGY
NS4A	1693–1708	Genotype 1 specific, coated antigen	AIIPDREVLYQEFDEM
NS4A	1693–1708	Genotype 2 specific, coated antigen	VVAPDKEVLYEAFDEM
Core‐NS4A chimera		Genotype 1 specific, competitive antigen	KARRPEGRTWAQPGYAIIPDREVLYQEFDEM
Core‐NS4A chimera		Genotype 2 specific, competitive antigen	KDRRTTGKSWGRPGYVVAPDKEVLYEAFDEM

^aRelative amino acid numbering according to the HCV strain H77 (GenBank No: AF009606) 21.

Limit of Detection (LOD) of the Serotyping Assay

Limit of detection was defined as the lowest anti‐HCV S/Co value at which the HCV serotype could still be identified with a probability of ≥95% using the serotyping assay. Two pooled serum samples were prepared by mixing 10 samples with HCV genotype 1 and 10 samples with genotype 2 from the CCgenos study, respectively. Both samples were serially diluted using negative human sera, and then the S/Co values were measured using the Vitros anti‐HCV assay (Ortho‐Clinical Diagnostics, High Wycombe, UK). At each S/Co level, twenty aliquots were serotyped. LOD was calculated by Probit analysis.

Quality Controls

Four quality controls with HCV genotype 1, 2, 3, and 6 were included in each run. They were pooled serum samples of the same genotype and diluted with the negative sera until the anti‐HCV levels approached two‐fold LOD. All the controls must generate the correct serotyping results before the results could be reported.

Subjects and Samples

The study was approved through the Peking University People's Hospital Ethical Committees (20120606). All the participants provided the written consent.

A total of 997 Chinese CHC patients were enrolled in a nationwide cross‐sectional study, the CCgenos study 22. The clinical characteristics of the patients such as the HCV genotype and RNA were summarized in our previous study 22. The samples were genotyped originally using the Versant HCV genotype 2.0 assay (LiPA 2.0). The anti‐HCV antibodies were tested using the Vitros anti‐HCV assay. The consistent genotype result yielded by the LiPA 2.0 assay and by the serotyping assay was considered as the confirmed result, whereas the inconsistent results should be further confirmed by the reference genotyping method, i.e., the sequence analysis of the nonstructural (NS) 5B region or the 5′‐untranslated region (UTR). The procedurals of the LiPA 2.0 assay and the reference genotyping assay were described in detail in our previous study 14. In the subsequent CCgenos follow‐up study, some patients achieved SVR after peginterferon treatment. Their serum samples after SVR were collected and serotyped as well, and the paired serotyping results were compared.

Serum samples from our two follow‐up cohorts were also serotyped using the novel assay. One cohort was from a long‐term follow‐up cohort in the Guan County, Hebei Province 23, and the other cohort was from the Dingzhou County, Hebei province. These subjects were all paid plasma donors. Patients without enough serum for serotyping were not included. Totally, 326 samples were serotyped, among which 260 samples were from CHC patients and the remaining 66 samples were from people with SVC. These people were defined as with SVC. First, they had undetectable serum HCV RNA using the Abbott RealTime HCV RNA assay with an LOD of 12 IU/ml. Second, they had exposed to HCV and had repeated positive anti‐HCV results using the local anti‐HCV screening assays in the earlier period.

Statistics

Differences in the categorical variables were analyzed using the chi square test or the Fisher's exact test as appropriate. Differences in the means of continuous variables following the normal distribution were tested using the unpaired t‐test. Statistics were performed using the GraphPad Prism version 6.02 (GraphPad Software Inc., San Diego, CA). Statistical significance was defined as P < 0.05.

Results

The Interpretation of the HCV Serotyping Results

A reactive non‐neutralized well was defined as OD ≥0.20. A reactive genotype‐specific well was defined according to the formula: OD_{genotype‐specific well}−OD_{completely neutralized well} ≥0.20 × OD_{non‐neutralized well}. A reactive non‐neutralized well plus a reactive genotype‐specific well or both reactive wells indicated the corresponding genotype(s) result. A non‐reactive non‐neutralized well without any reactive genotype‐specific wells indicated a non‐genotype 1 or 2 result. A reactive non‐neutralized well but without any reactive genotype‐specific wells, or any other patterns of the reactivity indicated an indeterminate result.

LOD of the Serotyping Assay

Limit of detection of the serotyping assay was 2.34 S/Co [95% confidence interval (CI): 1.86 S/Co‐3.97 S/Co] for genotype 1, and 2.22 S/Co (95% CI: 1.60 S/Co‐3.00 S/Co) for genotype 2.

Comparison of the HCV Genotyping and Serotyping Results of the Samples From the CCgenos Study

Totally 958 samples were available for the serotyping assay and had confirmatory genotyping results as well (Fig. 1). All these samples had high positive anti‐HCV results, ranging from 12.06 S/Co to 36.55 S/Co, among which 929 samples generated definite serotype results. The remaining 29 samples yielded indeterminate results and were consequently excluded from the further comparison (Table 2). The overall consistency between the genotyping and serotyping assays was 91.50% (850/929, 95% CI: 89.53−93.13%). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for different genotypes are shown in Table 3.

Figure 1.

Testing sequence and results of the samples from the CCgenos Study (n = 997).

Table 2.

Overall comparison of the HCV serotyping and genotyping results (n = 958)

HCV serotyping assay	LiPA 2.0 or direct sequencing assay
	GT 1	GT 2	GT 1/2	GT 3	GT 6	Total
GT 1	506	0	0	3	3	512
GT 2	10	218	0	6	10	244
GT 1/2	0	0	2	0	0	2
Non‐GT 1 or 2	35	12	0	83	41	171
Indeterminate	19	2	0	2	6	29
Total	570	232	2	94	60	958

GT, genotype. The bold indicates the number of the samples with consistent results.

Table 3.

Performance characteristics of the serotyping assay for different genotypes (n = 958)

Performance characteristics	GT 1	GT 2	GT ½ mixture	Non‐GT 1 or 2
Specificity (%, with 95% CI)	98.45 (96.49–99.37)	96.42 (94.72–97.60)	100 (99.50–100.00)	94.15 (92.24–95.63)
Sensitivity (%, with 95% CI)	88.77 (85.82–91.19)	93.97 (89.87–96.53)	100 (17.79–100.00)	80.52 (73.20–86.28)
PPV (%, with 95% CI)	98.83 (97.34–99.52)	89.34 (86.61–92.79)	100 (19.79–100.00)	72.51 (65.08–78.79)
NPV (%, with 95% CI)	85.65 (81.97–88.70)	98.04 (96.65–98.88)	100 (99.50–100.00)	96.19 (94.54–97.37)

CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value.

Serotyping Results for the Samples With Inconclusive Genotyping Results Using the Molecular Assays

Ten samples from the CCgenos study failed to be genotyped or yielded inconclusive genotyping results using the molecular genotyping assays (Fig. 1). They had low level of HCV RNA ranging from 57 to 390 IU/ml while high titer of anti‐HCV ranging from S/Co 14.20 to 27.80. These samples were all successfully serotyped. Seven were serotyped as genotype 1, and the remaining three were serotyped as genotype 2. To verify the serotyping results, we also serotyped the samples using another assay, the Sysmex HISCL HCV serotyping assay (Sysmex Corporation, Kobe, Japan). It was an automatic commercial assay based on the chemiluminescent enzyme immunoassay technology. The consistency between the two assays was 100% (10/10).

Comparison of the Paired Serotyping Results Before and After Treatment

Hundred and ninety paired samples from patients achieving SVR were analyzed. The baseline serotyping results were confirmed using the molecular methods. After antiviral treatment, 181 or 95.26% (95% IC: 91.24–97.49%) samples still generated concordant serotyping results. The remaining six genotype‐1 samples and three genotype‐2 samples generated inconsistent or indeterminate results (Table 4).

Table 4.

Comparison of the paired serotyping results (n = 190)

After treatment	Before treatment
	GT 1	GT 2	Non‐GT 1/2	Total
GT 1	104	0	0	104
GT 2	0	52	0	52
Non‐GT 1/2	2	2	25	29
Indeterminate	4	1	0	5
Total	110	55	25	190

The bold indicates the number of the samples with consistent serotyping results before and after therapy.

Comparison of the Distribution of HCV Genotypes in CHC Patients and in Individuals With SVC

Sixty‐six out of 326 individuals spontaneously cleared the HCV. The SVC rate was 20.25%. The characteristics of the CHC patients and the SVC individuals are shown in Table 5. There was no difference in the genotype distribution between the CHC and SVC groups (P = 0.08).

Table 5.

The characteristics of the CHC patients and the individuals with SVC (n = 326)

Variables	Total, n = 326	Individuals with SVC, n = 66	CHC patients, n = 260	P‐value
Age, years mean ± SD	47.85 ± 16.40	48.1 ± 20.51	44.40 ± 17.90	0.15
Gender
Male	206 (63.19%)	36 (54.55%)	170(65.38%)	0.12
Female	120 (36.81%)	30 (45.45%)	90 (34.62%)
Duration of infection (y)	22.00 ± 7.09	22.69 ± 13.09	21.44 ± 7.92	0.46
Anti‐HCV titer (by the Vitros assay, S/Co)	25.67 ± 6.34	12.30 ± 9.45	28.30 ± 7.44	<0.01
HCV serotype
1	237(72.70%)	44 (66.67%)	193(74.23%)	0.08
2	62(19.02%)	12 (18.18%)	50(19.23%)
1/2 mixture	16(4.91%)	3 (4.55%)	13(5.00%)
Non‐genotype 1 or 2	2(0.61%)	2 (3.03%)	0
Indeterminate	9(2.76%)	5 (7.58%)	4a(1.54%)
IL28 SNP (rs12979860)
CC	284 (87.11%)	59(89.39%)	225(86.54%)	0.68
CT	42 (12.89%)	7(10.61%)	35(13.46%)
TT	0	0	0

^aTwo samples generated genotype‐1 and two samples generated genotype‐2 results using the sequence analysis method.

Discussion

As compared to the molecular genotyping assays, the novel serotyping assay is a simple and while‐you‐wait testing, taking less than 1 hr to generate results. Besides, it seldom suffers from the PCR carryover contamination and is less dependent of special physical facilities. Moreover, the interpretation of the serotyping result is easier than that of the phylogenetic analysis of the viral sequences. Therefore, it is suitable for most of the clinical laboratories, especially for those in the resource‐limited areas.

The samples for the serotyping assay should be anti‐HCV positive. First, we assessed the lower limit of detection of the assay. It could successively serotype the sample with low level of anti‐HCV antibodies (2.00 S/Co‐3.00 S/Co by the Vitros anti‐HCV assay). In clinical settings, the majority of the CHC patients had high titer of anti‐HCV antibodies (≥8.00 S/Co by the Vitros anti‐HCV assay) 24. All the samples from our CCgenos study had anti‐HCV results above 12.00 S/Co. Samples from the SVC individuals, however, had lower level of antibodies, ranging from 3.04 S/Co to 18.31 S/Co, but the serotyping assay was analytically sensitive enough to detect these samples.

We evaluated the performance of the serotyping assay in a large CHC population. The overall consistency between the serotyping assay and the molecular genotyping assay was 91.50%. The specificity and sensitivity of the assay for determining HCV genotypes 1, 2, and 1/2 mixture were comparable or superior to those of other serotyping assays such as the Murex ELISA assay and the Chiron RIBA strip immunoblot assay 25, 26, 27, 28, 29, 30, 31, although we did not perform the head‐to‐head comparisons. Two methodological modifications may contribute to the performance improvement. First, both NS4 and core antigen fragments are included in the novel assay. The NS4 region was the first choice for developing HCV serotyping assays, because the N‐terminal part of the protein is heterogeneous for different genotypes. But the sensitivity (i.e., the genotype detection rate) was limited (approximately 80%), especially for the immunocompromised population 28. The core antigen was also included in some assays 31, but the specificity was low because the core sequence was conserved. Combination of the two proteins was helpful for the improvement of the specificity and sensitivity. Second, this assay is based on the competitive inhibition ELISA principle, which helped to reduce the interference and improve the specificity.

Twenty nine samples out of 958 CCgenos samples (3.03%) generated indeterminate serotyping results. They had comparable titers of anti‐HCV antibodies with those with definite serotyping results (26.10 ± 7.75 S/Co vs. 26.67 ± 4.11 S/Co, P = 0.70). Therefore, the indeterminate serotyping results for the CHC patients may be caused by the non‐reactivity between the serotype‐specific antigens and the serum anti‐HCV antibodies, but not by the low level of anti‐HCV antibodies.

It is important to evaluate an assay in the context of its intended use. The serotyping assay was not intended for use for identifying the exact genotype except genotypes 1 and 2. It can be used as a genotype screening assay because it is simple, rapid and cheap, especially suitable for use in areas where the genotypes 1 and 2 are predominant. Samples with a “non‐GT 1 or 2” result (probably genotype 3 in most countries) or an indeterminate result should undergo further molecular genotyping for antiviral therapy using the peginterferon or DAAs.

When the samples are from the area where the genotypes 3 to 6 are prevalent, the serological cross‐reaction may be a potential issue. For this reason, we further evaluated the specificity of the serotyping assay for detecting non‐genotype 1 or 2 samples as well. In China, genotype ‐4 or ‐5 HCV infected patients have been never reported 22. Therefore, the non‐genotype 1 or 2 belonged to genotype 3 or 6. The performance for genotype‐3 samples was similar with that for genotype 1, genotype 2, or 1/2 mixture; whereas the performance for detecting genotype 6 was barely satisfactory. In 60 genotype‐6 samples, only 41 samples (68.33%) were correctly identified as non‐genotype 1 or 2 using the serotyping assay. Three samples were misclassified as genotype 1; ten samples were misclassified as genotype 2; and the remaining six samples yielded indeterminate results. The cross‐reaction between the peptides and anti‐HCV antibodies might result in the misclassification. It is conceivable because even the molecular genotyping tests have the challenge in differentiating genotype 6 from genotype 1 occasionally due to the sequence similarity between the two genotypes 14. We also found the misclassification of genotype 6 into genotype 2. Reverse transcription‐PCR and sequence analysis, however, were not performed to confirm the sequence similarity due to the limited sample volume. Fortunately, the misclassification was mainly found in samples with genotype 6. For samples with other genotypes, the specificity was high and few misclassified samples were found. In the CCgenos study, we enrolled the patients proportionally according to the population size of each province. Therefore, the percentage 6.3% reflected the real‐world distribution of genotype‐6 infected patients in China 22. The accuracy of this new serotyping assay was 91.50%, but it can be supposed that with the increased percentage of genotype 6, the accuracy of the HCV serotyping would decrease. Genotype 6 is only prevalent in South China and Southeast Asian countries 22; higher prevalence of genotype 6 has also been reported in certain populations such as thalassemia major patients and drug users 32. Due to the relatively low specificity and sensitivity for detecting genotype 6, the serotyping assay is recommended to be used neither in such special populations nor in areas where the genotype 6 is endemic. Moreover, the serotyping assay is not recommended to be used where the HCV of genotypes 4 or 5 is endemic 3, either, because we did not evaluate the performance of the assay for these genotypes.

The advantage of the serotyping assay is that it can determine the genotype of the CHC patients carrying low level of serum HCV RNA. Usually, the samples of these patients cannot be genotyped or yielded ambiguous genotyping results using the molecular methods. In our study, 10 samples from the CCgenos study could not be genotyped but they were all accurately serotyped.

Another advantage of the serotyping assay is that it can identify the genotype of individuals who cleared the virus. Our results showed that the genotypes of CHC patients could still be accurately identified after achieving SVR. Moreover, research on the association between the viral genotype and SVC suffered from the unavailability of the genotypes by means of the molecular methods. The serotyping assay, however, provided us a tool to address the issue. Sixty‐six out of 326 individuals spontaneously cleared the HCV. The spontaneous viral clearance rate was similar as previous reported, ranging from 18–34% 33.Our result revealed that the percentages of the HCV serotypes in the population with and without SVC was not significantly different, which was consistent with previous studies 19, 20. On the other hand, there were different findings, where the patients with genotype‐1 HCV had less chance of spontaneous clearance 16, 17, 18. Three reasons might underlie the discrepancy. First, the host genotype, for example, the IL28B polymorphism, also contributes to the SVC 34. We found no difference in the IL28B polymorphism between the CHC and SVC groups, while the other studies failed to provide such information. Second, the transmission route and the exposure dose of HCV also play an important role on the spontaneous clearance 35. For our subjects, the transmission route was uniform (i.e., paid plasma donors); they were infected through high‐dose exposure, whereas in other studies the transmission route and the exposure dose were heterogeneous. Third, the subjects enrolled in our study had a longer duration since the exposure to HCV (22 years on average) than that in the previous studies. Some genotype‐1 patients might spontaneously clear the virus in the longer term after the acute phase of infection. Our finding partly explains why the distribution of the HCV genotypes generally keeps unchanged worldwide. Furthermore, our previous study showed that the genotype did not influence the long‐term outcome of the CHC, either 23.

Our conclusion on the relationship between HCV genotype and SVC, however, has some limitations. First, it was a retrospective analysis. There may be some individuals with SVC who further cleared the anti‐HCV antibodies 15, whereas only those with positive anti‐HCV could be serotyped. For the individuals who have spontaneously cleared the serum HCV RNA as well as the anti‐HCV antibodies, their HCV serological tests were all negative. Therefore, they could not be enrolled in our study. Second, patients experiencing a long history of HCV infection (more than 20 years) often carried low level of serum anti‐HCV. For the serotyping assay, the core and NS4 fragments were used to detect the HCV genotype‐specific antibodies. However, the titer of the serum antibodies against the core antigen gradually decreased with time, and the avidity of the antibodies against the NS4 protein specific for genotypes 1 and 2 also decreased 36. Consequently, the assay was more likely to yield indeterminate results in the SVC individuals although the levels of the samples were above the LOD of the serotyping assay. In our study, the proportion of these results accounted for 7.58% in SVC individuals while only 1.54% in CHC patients. This might affect the statistical analysis. Third, the serotyping assay yielded some non‐genotype 1 or 2 results due to its limited coverage for all the genotypes. We did not know the exact genotypes of these individuals. Meanwhile, limited number of subjects with genotypes other than genotypes 1 and 2 were enrolled. Hence, further investigations are warranted.

In conclusion, the novel HCV serotyping assay is accurate for the detection of HCV genotype 1, 2, and genotypes 1/2 mixture, providing a simple, cheap and while‐you‐waiting alternative. Therefore, it is particularly suitable for application in the resource‐limited settings. For the genotype‐3 to ‐6 samples, it cannot identify the exact genotype but yield result of non‐genotype 1 or 2. It is accurate for detecting genotype‐3 samples but not recommended to be used where the genotypes‐6 HCV infection is endemic due to the relatively low sensitivity and specificity. Furthermore, using the serotyping assay, the genotypes of people with SVC can be determined. Genotype distribution is similar between the CHC patients and the individuals with SVC. Therefore, HCV genotype may not play an important role during the SVC course.

Ethics Approval

The study was approved through the Peking University People's Hospital Ethical Committees (20120606).

Author Contributions

All the authors have accepted the responsibility for the entire content of this submitted article and approved submission.

Employment or Leadership

None declared.

Honorarium

None declared.