Kaohsiung Journal of Medical Sciences (2015) 31, 271e277
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ORIGINAL ARTICLE
Host and virological characteristics of patients with hepatitis C virus mixed genotype 1 and 2 infection Chung-Feng Huang a,b, Ching-I Huang a,c, Ming-Lun Yeh c, Jee-Fu Huang c,d, Ming-Yen Hsieh c, Zu-Yau Lin c,d, Shinn-Cherng Chen c,d, Ming-Lung Yu c,d, Chia-Yen Dai c,d,e,f,*, Wan-Long Chuang c,d a
Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan b Department of Occupational Medicine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan c Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan d Faculty of Internal Medicine, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan e Department of Occupational Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan f Department of Preventive Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Received 3 July 2014; accepted 6 February 2015
Available online 12 March 2015
KEYWORDS Genotype; Hepatitis C virus; Interleukin-28B; Mixed infection
Abstract The prevalence and characteristics of patients with hepatitis C virus mixed genotype 1 and 2 infection (HCV-1/2) remains unclear. For each HCV-1/2 patient with histological data available, two age- and sex-matched HCV-1 and HCV-2 infected patients were selected for comparison, respectively. Of the 2776 patients, 261 (9.4%) patients were identified as having mixed HCV-1/2 infection. The histological severity did not differ among HCV-1/2 patients and controls. The proportion of patients with the interleukin-28B (IL-28B) rs8099917 TT genotype did not differ between patients with mixed-1/2 and HCV-1 infection (82.6% vs. 86.5%, p Z 0.38). However, HCV-2 infected patients had a significantly higher proportion of the rs8099917 TT genotype compared to patients with mixed HCV-1/2 infections (91.6% vs. 82.6%, p Z 0.03). The HCV RNA levels were similar in patients with HCV-1/2 and HCV-1 infections (5.5 0.8 log IU/mL vs. 5.5 0.9 log IU/L, p Z 0.73), which were both significantly
Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, No. 100, Tzyou 1st Road, Kaohsiung 807, Taiwan. E-mail addresses: [email protected], [email protected] (C.-Y. Dai). http://dx.doi.org/10.1016/j.kjms.2015.02.004 1607-551X/Copyright ª 2015, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
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C.-F. Huang et al. higher than that of HCV-2 infection (5.1 0.9 log IU/mL, both p < 0.001). Linear regression analysis revealed that the factors of HCV-1/2 infection [b: 0.204; 95% confidence interval (CI): 0.183e0.598, p < 0.001], body weight (b: 0.150; CI: 0.003e0.021, p Z 0.008), and age (b: 0.139; CI: 0.020 to 0.002, p Z 0.01) were independently correlated with HCV RNA levels in patients with HCV-2 and mixed HCV-1/2 infection. We concluded that the host genetic and viral presentations of patients with mixed HCV-1/2 infection were more similar to those of patients with HCV-1 infection than patients with HCV-2 infection. Copyright ª 2015, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction Hepatitis C virus (HCV) infection is a major health issue that affects an estimated 170 million people worldwide [1,2]. An increased incidence of HCV-related end-stage liver disease and hepatocellular carcinoma has been noted in the past decade [3,4]. There are six HCV genotypes and more than 50 subtypes, which tend to segregate geographically. Regional distributions of HCV genotypes are diverse, reflecting the variability of acquisition routes and ethnic differences. HCV genotypes 1 (HCV-1), 2, and 3 have a fairly broad geographical distribution, whereas HCV genotypes 4, 5, and 6 are generally restricted to specific regions [5,6]. The understanding of viral genotype not only facilitates HCV treatment on an individualized basis [6e8] but also helps to elucidate the pathological nature of HCV infection [9]. For example, patients with HCV-1 infection have a lower sustained response (SVR) rate than patients with HCV-2. HCV-1b has been shown to have a higher risk for developing into hepatocellular carcinoma compared to other viral genotypes [9]. However, interleukin 28B (IL-28B) genetic variants have been recognized as the most important host factor either in the treatment of HCV infection [10e14] or in the influence of spontaneous HCV clearance [15]. We have demonstrated that viral genotype differences regarding IL-28B genotype distribution exist, which might in part be responsible for different treatment responses between HCV-1 and HCV-2 [16]. We have recently demonstrated that the treatment efficacy of patients with mixed HCV-1 and HCV-2 infection (HCV-1/2) was satisfactory in Asian patients receiving pegylated interferon/ribavirin combination therapy [17]. However, the prevalence of patients with mixed HCV-1/2 infection is unclear, and the clinical presentation, in terms of viral and host characteristics, such as IL-28B genetic variants, remains elusive. Taiwan possesses a unique epidemiological background because HCV is widely endemic and both HCV-1 and HCV-2 prevail throughout the entire country [6,18,19]. It is essential to fully clarify the host and virological characteristics of this population before the treatment strategy can be recommended. We herein retrospectively analyzed a hospital-based cohort with a respectable sample size to address this issue.
Materials and methods Eligible individuals were treatment-naı¨ve chronic hepatitis C patients that were enrolled consecutively in a medical
center and two core regional hospitals in southern Taiwan from 2001 to 2011. First, we analyzed the prevalence and characteristics of all the patients with mixed HCV-1/2 infection in the hospital cohort. To clarify the differences between mixed infections and other viral genotypes, patients with mixed HCV-1/2 infection with available histological data were selected to compare with age- and sexmatched HCV-1 and HCV-2 controls. All of the patients provided written informed consent prior to enrollment. Anti-HCV antibodies were detected using a thirdgeneration, commercially available enzyme-linked immunosorbent assay kit (AxSYM 3.0; Abbott Laboratories, Chicago, IL, USA). Serum HCV RNA was obtained using realtime polymerase chain reaction (COBAS AMPLICOR Hepatitis C Virus Test, ver. 2.0; Roche, Branchburg, NJ, USA; detection limit: 50 IU/mL). HCV genotypes were determined using the Okamoto method [20]. Liver histology was graded and staged according to the scoring system described by Knodell and Scheuer [21]. Biochemical items were measured on a multichannel auto analyzer (Hitachi Inc., Tokyo, Japan). The IL-28B genotypes of the patients were determined by the ABI TaqMan single nucleotide polymorphism (SNP) genotyping assays (Applied Biosystems, Foster City, CA, USA) using the predesigned commercial genotyping assays (ABI Assay ID: C__11710096_10). Briefly, polymerase chain reaction (PCR) primers and two allelespecific probes were designed to detect the specific SNP target. The PCR reactions were performed in 96-well microplates using ABI 7500 real-time PCR (Applied Biosystems). Allele discrimination was achieved by detecting fluorescence using ABI System SDS software version 1.2.3 (Applied Biosystems). The institutional review board at Kaohsiung Medical University Hospital approved the protocol, which conformed to the guidelines of the International Conference on Harmonization for Good Clinical Practice.
Statistical analyses IL-28B rs8099917 was selected as the candidate SNP analysis in the current study based on findings in our previous studies [10,11,22]. The frequency was compared between groups using the c2 test, with the Yates correction or Fisher’s exact test. Group means, presented as the mean values and standard deviation, were compared using analysis of variance and the Student t test or the ManneWhitney U test. Serum HCV RNA levels were expressed after logarithmic transformation of original values. To represent the severity of liver fibrosis, the aspartate
HCV mixed infection
273
aminotransferase (AST)-to-platelet ratio index (APRI) was calculated using the following equation: (AST level/upper limit of normal range)/platelet counts (109/L) 100 [23,24]. The frequencies of the rare allele of the rs8099917 genotype were too low; thus, the rare homozygote and heterozygote were combined together while analyzing the SNP. Logistic regression and linear regression analysis were performed to evaluate the independent factors associated with HCV RNA levels. The statistical analyses were performed using the SPSS 12.0 statistical package (SPSS, Chicago, IL, USA). All statistical analyses were based on twotailed hypothesis tests with a significance level of p < 0.05.
Results Overall patient characteristics in the hospitalbased cohort Of the 2776 patients, 311 (11.2%) were identified with HCV mixed infection and 261 (9.4%) were identified with mixed HCV-1/2 infections (Fig. 1). The basic demographic, virological, and clinical features of the 261 patients are shown in Table 1. The mean age was 51.0 11.9 years. Males accounted for 59.4% of the population. The mean HCV RNA levels were 5.7 0.9 log IU/mL. Among the 184 patients with available IL-28B genotype information, the rs8099917 TT genotype accounted for 154 (83.7%) patients, and 30 (16.3%) patients had the rs8099917 non-TT genotype. The majority of mixed HCV infections in patients were of the 1b/2a genotype (44.8%), followed by the 1b/2b genotype (43.7%). The clinical presentations between the two subpopulations were identical except that patients with HCV-1b/2b infection were younger and had higher HCV RNA levels (Table S2).
Characteristics of mixed HCV-1/2 infection in comparison with other HCV genotypes A total of 104 patients had histological data available. The characteristics did not differ between the patients with or without available histology data except that patients with
Table 1 Basic demographic and clinical features of the 261 patients with mixed HCV-1 and HCV-2 infection. Sex, n (%) Male Female Age (y), mean SD Body weight (kg), mean SD Rs8099917 TT genotype, n/N (%)a Baseline HCV RNA (log IU/mL), mean SD Baseline viral loads > 400,000 IU/mL, n (%) Fibrosis, n (%) 0e2 3e4 HAI, mean SD AST (IU/L), mean SD ALT (IU/L), mean SD GGT (IU/L), mean SD APRI, mean SD Fasting sugar (mg/dL), mean SD Genotype distribution, n (%) 1a þ 2a 1b þ 2a 1b þ 2b 1a þ 2a þ 2b 1a þ 1b þ 2a þ 2b 1a þ 1b þ 2a
155 (59.4) 106 (40.6) 51.0 11.9 67.4 12.9 154/184 (83.7) 5.7 0.9 157 (60.2) 76 (73.1) 28 (26.9) 5.1 2.7 94.4 68.0 144.3 154.8 76.4 78.5 2.6 2.1 102.1 34.8 2 (0.8) 117 (44.8) 114 (43.7) 26 (10.0) 1 (0.4) 1 (0.4)
ALT Z aspartate aminotransferase; APRI Z alanine aminotransferase to platelet ratio index; AST Z alanine aminotransferase; GGT Z gamma-glutamyl transferase; HAI Z histological activity index; HCV Z hepatitis C virus; SD Z standard deviation. a Data available in 184 patients.
unavailable data were older and had lower AST levels (Table S1). To fully explore the characteristics of patients with mixed HCV-1/2 infection, patients that were age- and sex-matched and infected with either HCV-1 or HCV-2 were selected for comparison with 1:2 ratios (Fig. 1). As shown in Table 2, the histological severity in terms of liver fibrosis and histological activity index did not differ among patients
2776 treatment-naïve CHC patients
Pure HCV-1 (n = 1294, 46.6%)
Pure HCV-2 (n = 1162, 41.9% )
Mixed HCV genotypes (n = 311, 11.2%)
Other genotypes (n = 9, 0.3%)
Mixed other genotypes (n = 50,1.8%)
208 age-, sex-matched HCV-1 and HCV-2 controls, respectively
Figure 1.
Histology available (n = 104)
Histology unavailable (n = 157)
Distribution of HCV genotypes in the cohort. CHC Z chronic hepatitis C; HCV Z hepatitis C virus.
274 Table 2
C.-F. Huang et al. Basic demographic and clinical features of the three patient groups with different HCV genotypes.
Sex, n (%) Male Female Age (y), mean SD Body weight (kg), mean SD Rs8099917 TT genotype, n/N (%) a Fibrosis, n (%) 0e2 3e4 HAI, mean SD Baseline HCV RNA (log IU/mL), mean SD AST (IU/L), mean SD ALT (IU/L), mean SD GGT (IU/L), mean SD APRI, mean SD Fasting sugar (mg/dL), mean SD
Mixed HCV-1/2 (n Z 104)
HCV-1 (n Z 208)
HCV-2 (n Z 208)
64 (61.5) 40 (38.5) 48.8 12.1 66.3 11.8 71/86 (82.6) *
125 (60.1) 83 (39.9) 48.5 11.4 65.8 10.2 180/208 (86.5)
128 (61.5) 80 (38.5) 47.9 10.8 65.5 11.3 174/190 (91.6) *
76 (73.1) 28 (26.9) 5.1 2.7 5.5 0.8 **
152 (73.4) 55 (26.6) 4.8 2.6 5.5 0.9 **
160 (77.7) 46 (22.3) 5.0 2.7 5.1 1.0 **
105.1 64.6 158.6 109.5 77.2 70.6 2.9 2.2 105.8 46.0
97.8 59.3 149.8 87.9 60.9 50.6 2.7 2.0 100.9 26.3
103.2 65.6 167.0 110.3 65.9 57.8 2.9 2.5 102.8 39.2
p 0.94
0.78 0.82 0.08 0.31
0.7 < 0.001 ** 0.54 0.23 0.07 0.51 0.54
* p Z 0.03; ** p < 0.001. ALT Z aspartate aminotransferase; APRI Z alanine aminotransferase to platelet ratio index; AST Z alanine aminotransferase; GGT Z gamma-glutamyl transferase; HAI Z histological activity index; HCV Z hepatitis C virus; mixed HCV-1/2 genotype Z HCV genotype 1 and HCV genotype 2 mixed infection; SD Z standard deviation. a Data available in 484 patients.
with mixed HCV-1/2, HCV-1, and HCV-2 infection. The proportion of the IL-28B rs8099917 TT genotype did not differ between patients with mixed-1/2 and HCV-1 infection (82.6% vs. 86.5%, p Z 0.38). However, HCV-2 infected patients had a significantly higher proportion of the rs8099917 TT genotype compared to patients with mixed HCV-1/2 infection (91.6% vs. 82.6%, p Z 0.03). The HCV RNA levels were similar between patients with HCV-1/2 and
Table 3
HCV-1 infection (5.5 0.8 log IU/mL vs. 5.5 0.9 log IU/L, p Z 0.73), which were both significantly higher than that of HCV-2 infection (5.1 0.9 log IU/mL, both p < 0.001). The HCV RNA levels were so similar between patients with mixed HCV-1/2 and HCV-1 infection that we further analyzed the characteristics associated with HCV RNA levels among patients with HCV-2 and mixed HCV-1/2 infection. As shown in Table 3, younger age, higher body
Factors associated with high or low viral loads among patients with HCV-2 and mixed HCV-1/2 infection. High viral loads (n Z 130)
Male sex, n (%) Age (y), mean SD Rs8099917 TT genotype, n/N (%) b Fibrosis 3/4, n/N (%) HAI, mean SD Body weight (kg), mean SD AST (IU/L), mean SD ALT (IU/L), mean SD GGT (IU/L), mean SD APRI, mean SD Fasting sugar (mg/dL), mean SD HCV genotype, n (%) HCV-2 Mixed HCV-1/2
Low viral loads (n Z 182)
86 (66.2) 46.5 12.8 99/115 (86.1) 31/130 (23.8) 5.1 2.8 68.1 11.4 100.0 64.8 165.4 119.4 72.1 59.8 2.6 2.2 103.7 31.8
106 (58.2) 49.5 9.9 146/161 (90.7) 43/180 (26.4) 4.8 2.6 64.1 11.1 106.6 65.5 163.3 103.0 67.8 64.4 3.1 2.5 103.9 47.6
73 (56.2) 57 (43.8)
135 (74.2) 47 (25.8)
p
0.16 0.03 0.23 0.99 0.12 0.002 0.38 0.87 0.55 0.06 0.96 0.001
a
Logistic regression analysis of factors associated with high viral loads OR
95% CI
p
0.979
0.958e1.000
1.030
1.008e1.052
0.007
2.171
1.324e3.559
0.002
0.05
ALT Z aspartate aminotransferase; APRI Z alanine aminotransferase to platelet ratio index; AST Z alanine aminotransferase; CI Z confidence interval; GGT Z gamma-glutamyl transferase; HAI Z histological activity index; HCV Z hepatitis C virus; mixed HCV-1/ 2 Z HCV genotype 1 and HCV genotype 2 mixed infection; OR Z odds ratio; SD Z standard deviation. a High viral loads defined as HCV RNA > 400,000 IU/Ml. b Data available in 276 patients.
HCV mixed infection
275
Table 4 Linear regression analysis of factors associated with viral loads among patients with HCV-2 and mixed HCV1/2 infection. a b Body 0.012 weight Mixed 0.391 HCV-1/2 genotype Age 0.011
Standard error
95% CI for b
b
p
0.004
0.003
0.021
0.150
0.105
0.183
0.598
0.204 < 0.001
0.004
0.020 0.002 0.139
0.008
0.01
CI Z confidence interval; HCV Z hepatitis C virus; mixed HCV1/2 genotype Z HCV genotype 1 and HCV genotype 2 mixed infection. a Odds ratios (ORs) are for body weight (per 1 kg increase), age (per 1 year increase), and HCV genotype (mixed HCV genotype 1and 2 vs. HCV genotype 2).
weight, and mixed HCV-1/2 infection were factors associated with high viral loads, defined as HCV RNA levels > 400,000 IU/mL, in univariate analysis. Multivariate analysis revealed that the strongest factor associated with high viral loads was mixed HCV-1/2 infection [odds ratio (OR)/95% confidence interval (CI): 2.171/1.324e3.559, p Z 0.002] followed by body weight (OR/CI: 1.030/1.008e1.052, p Z 0.007), and age (OR/CI: 0.979/0.958e1.000, p Z 0.05). Linear regression analysis revealed that the factors of HCV1/2 infection (b: 0.204; 95% CI: 0.183e0.598, p < 0.001), body weight (b: 0.150; 95% CI: 0.003e0.021, p Z 0.008) and age (b: 0.139; 95% CI: 0.020e0.002, p Z 0.01) were independently correlated with HCV RNA levels (Table 4).
The interplay of HCV RNA levels and host IL-28B genotype As shown in Table 5, the HCV RNA levels did not differ between HCV-1 patients with the rs8099917 TT genotype and the rs8099917 non-TT genotype (5.49 0.94 log IU/mL vs. 5.51 0.70 log IU/mL, p Z 0.92). The HCV RNA levels also did not differ between mixed HCV-1/2 patients with the rs8099917 TT genotype and the rs8099917 non-TT genotype (5.60 0.83 log IU/mL vs. 5.55 0.74 log IU/mL, p Z 0.92). However, the HCV RNA levels were significantly lower in HCV-2 patients with the rs8099917 TT genotype compared with those with the rs8099917 non-TT genotype (5.07 0.95 log IU/mL vs. 5.57 0.54 log IU/mL,
Table 5
p Z 0.003). The difference in HCV RNA levels between HCV2 and other HCV genotypes existed only in patients with the rs8099917 TT genotype (p < 0.001) but not in those with the rs8099917 non-TT genotype (Table 5). Among patients with the rs8099917 TT genotype, patients with mixed HCV-1/2 infection had a significantly higher proportion of high viral loads compared to patients with HCV-2 infection (59.2% vs. 33.9%, p < 0.001), and the proportion of high viral loads did not differ between non-TT genotype carriers with HCV-2 and mixed HCV-1/2 infection (53.3% vs. 50.0%, p Z 1) (Fig. 2).
Discussion To our knowledge, this is the first study to address the prevalence and characteristics of patients with mixed infections with HCV-1 and HCV-2. We demonstrated that one out of 10 HCV patients was infected with mixed HCV-1/2 in Taiwan. The liver disease severity, in terms of necroinflammation and fibrosis, was similar between the study population and those infected with other viral genotypes. Nevertheless, the host IL-28B genetic predisposition and viral presentation of patients with mixed HCV-1/2 infection were more similar to those with HCV-1 infection. Compared with patients with HCV-2 infection, patients with mixed HCV-1/2 infection had a significantly lower proportion of favorable IL-28B genotype carriage and higher HCV RNA levels. Superinfection of heterologous HCV in the same host was first demonstrated in chimpanzees [25,26]. Presentation as acute hepatitis in humans was reported shortly thereafter in early 1990 [27] and was further confirmed in a subsequent case-series study [28]. The definition of mixed HCV genotype infections is flexible and complex, as they might be comprised of different major genotypes or subtypes. The prevalence of mixed HCV genotype infections is also difficult to ascertain, with ranges from 0% to 25% having been reported depending on the genotyping method, definition of mixed infection, and study population [29e37]. The presence of mixed infections has been proposed as a potential cause for poor response to interferon-based antiviral therapy, as selection of minority genotype populations more refractory to antiviral treatment can occur during treatment [38]. The precision of studies on the transmission of HCV also depends on whether coexisting genotypes can be tracked along with the other ones. In the current study, we investigated patients with mixed infections of HCV-1 and HCV-2 for two reasons: first, the
HCV RNA levels in different HCV genotypes stratified by IL-28B genotype. Rs8099917 TT genotype
HCV-1 and mixed HCV-1/2 (log IU/mL), mean (SD) Mixed HCV-1/2 (log IU/mL), mean (SD) HCV-1 (log IU/mL), mean (SD) HCV-2 (log IU/mL), mean (SD)
5.50 5.60 5.46 4.99
(0.94) * (0.83) (0.97) * (1.02) *
Rs8099917 non-TT genotype 5.53 5.55 5.51 5.57
(0.70) (0.74) (0.70) (0.54)
p 0.87 0.84 0.80 0.003
* p < 0.001. HCV Z hepatitis C virus; IL-28B Z interleukin 28B; mixed HCV-1/2 Z HCV genotype 1 and HCV genotype 2 mixed infection; SD Z standard deviation.
276
Figure 2. Percentage of patients with high HCV RNA levels between patients with mixed HCV-1/2 and HCV-2 infection. The black bar represents patients with mixed HCV-1/2 infection. The white bar represents patients with HCV-2 infection. HCV Z hepatitis C virus; HVL Z high viral loads, defined as HCV RNA levels >400,000 IU/mL.
patients might be frequently encountered in daily practice in countries where both HCV genotypes prevail. Second, treatment strategies toward the two genotypes are largely different. It is therefore more rational and very essential to explore the characteristics of the virus and patients before we can fully address the treatment efficacy in this specific population. It has been suggested that patients infected with HCV-1 have higher HCV RNA levels compared with patients with HCV-2 infection [39]. Intriguingly, we demonstrated that patients with mixed HCV-1/2 infection had similar HCV RNA levels to patients with nonmixed HCV-1 but significantly higher HCV RNA levels than those with HCV-2 infection. Higher body weight was also independently associated with high viral loads. The precise reason was uncertain. Metabolic disarrangement in terms of insulin resistance and lipid disorders have been associated with HCV viral loads [39], which might be partially responsible for the result. Host IL-28B genetic variants are currently the most important host factor in determining viral eradication either by antiviral therapy [10e14] or self-limited HCV infection [15]. The distribution of IL-28B genetic variants would account for diverse treatment outcomes not only across different ethnicities of patients but also different viral genotypes [16]. We have previously demonstrated that the favorable IL-28B rs8099917 TT genotype was overrepresented among HCV-non-1 infected patients, compared with HCV-1 patients (91.4% vs. 85.0%, p Z 0.002) [16]. In the current study, we noticed that IL-28B genetic distribution in patients with mixed HCV-1/2 infection was more similar to patients with HCV-1 infection, with a lower proportion of favorable genotype carriage. Further studies to explore the treatment efficacy of the population based on these findings are warranted. Although patients were stratified according to IL-28B genotype, lower HCV RNA levels were only noted in HCV-2 patients with the favorable rs8099917 TT genotype. It has been suggested that HCV-1 patients with a favorable IL-28B genotype have higher HCV RNA levels [12], which might attribute to viral selection during evolution. However, the
C.-F. Huang et al. results were not consistent across all studies with different HCV genotypes [22,40]. The conflicting finding might in part result from selection bias of hospital-based studies. Further community-based studies enrolling larger sample size might be needed before a definite conclusion of the issue could be made. The limitation of the current study was that we failed to determine the predominant viral genotype in a given patient. To address this issue, a detailed cloning or deep sequence analysis might provide more precise evaluation. However, it is complicated and time-consuming and therefore is difficult to be applied to large-scale studies. Since the acquisition of HCV is frequently asymptomatic, we failed to judge the impact of duration of infection on liver fibrosis among study groups. The viral interference of heterologous HCV genotypes in the same host might be dynamic during the course of superinfection [18,28]. However, patients selected in the current hospital cohort were chronically infected and had been scheduled to receive antiviral therapy, so the viral presentation of mixed HCV1/2 infection in the same host was consistent in the current patient cohort. In conclusion, patients with mixed HCV-1/2 infection are frequently encountered in Taiwan. This should also be the case in areas where both viral genotypes are endemic. Both the viral and host genetic features in patients with mixed HCV-1/2 infection were more similar to those with HCV-1 infection than those with HCV-2 infection. In the era of direct antiviral agents (DAAs), so far, there is no solution for how to manage this specific population, unless pangenotypic DAAs are made widely available. The current study might provide insight and further guidance regarding the treatment of patients with mixed infections of HCV-1 and HCV-2.
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24 weeks in patients with genotype 2 chronic hepatitis C. Gut 2007;56:553e9. Raimondi S, Bruno S, Mondelli MU, Maisonneuve P. Hepatitis C virus genotype 1b as a risk factor for hepatocellular carcinoma development: a meta-analysis. J Hepatol 2009;50:1142e54. Huang CF, Huang JF, Yang JF, Hsieh MY, Lin ZY, Chen SC, et al. Interleukin-28B genetic variants in identification of hepatitis C virus genotype 1 patients responding to 24 weeks peginterferon/ribavirin. J Hepatol 2012;56:34e40. Huang CF, Yeh ML, Huang JF, Yang JF, Hsieh MY, Lin ZY, et al. Host interleukin-28B genetic variants versus viral kinetics in determining responses to standard-of-care for Asians with hepatitis C genotype 1. Antiviral Res 2012;93:239e44. Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009;461: 399e401. Suppiah V, Moldovan M, Ahlenstiel G, Berg T, Weltman M, Abate ML, et al. IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet 2009;41:1100e4. Tanaka Y, Nishida N, Sugiyama M, Kurosaki M, Matsuura K, Sakamoto N, et al. Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat Genet 2009;41:1105e9. Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O’Huigin C, et al. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 2009;461:798e801. Huang CF, Dai CY, Huang JF, Chuang WL, Yu ML. Linkage of the hepatitis C virus genotype and interleukin-28B genetic polymorphisms in Asian patients. Hepatology 2011;53:367e8. Huang CI, Huang CF, Huang JF, Dai CY, Yeh ML, Hsieh MY, et al. The treatment efficacy of pegylated interferon plus ribavirin therapy in chronic hepatitis C patients with mixed genotype 1/2 infection. J Gastroenterol Hepatol 2014;29:1012e8. Yang JF, Lin CI, Huang JF, Dai CY, Lin WY, Ho CK, et al. Viral hepatitis infections in southern Taiwan: A multicenter community-based study. Kaohsiung J Med Sci 2010;26:461e9. Chuang WL, Yu ML, Dai CY, Chang WY. Treatment of chronic hepatitis C in southern Taiwan. Intervirology 2006;49:99e106. Okamoto H, Tokita H, Sakamoto M, Horikita M, Kojima M, Iizuka H, et al. Characterization of the genomic sequence of type V (or 3a) hepatitis C virus isolates and PCR primers for specific detection. J Gen Virol 1993;74(Pt 11):2385e90. Scheuer PJ. Classification of chronic viral hepatitis: a need for reassessment. J Hepatol 1991;13:372e4. Yu ML, Huang CF, Huang JF, Chang NC, Yang JF, Lin ZY, et al. Role of interleukin-28B polymorphisms in the treatment of hepatitis C virus genotype 2 infection in Asian patients. Hepatology 2011;53:7e13. Lin ZH, Xin YN, Dong QJ, Wang Q, Jiang XJ, Zhan SH, et al. Performance of the aspartate aminotransferase-to-platelet ratio index for the staging of hepatitis C-related fibrosis: an updated meta-analysis. Hepatology 2011;53:726e36. Martinez SM, Fernandez-Varo G, Gonzalez P, Sampson E, Bruguera M, Navasa M, et al. Assessment of liver fibrosis before and after antiviral therapy by different serum marker panels in patients with chronic hepatitis C. Aliment Pharmacol Ther 2011;33:138e48. Prince AM, Brotman B, Huima T, Pascual D, Jaffery M, Inchauspe G. Immunity in hepatitis C infection. J Infect Dis 1992;165:438e43.
277 [26] Farci P, Alter HJ, Govindarajan S, Wong DC, Engle R, Lesniewski RR, et al. Lack of protective immunity against reinfection with hepatitis C virus. Science 1992;258:135e40. [27] Kao JH, Chen PJ, Lai MY, Chen DS. Superinfection of heterologous hepatitis C virus in a patient with chronic type C hepatitis. Gastroenterology 1993;105:583e7. [28] Kao JH, Chen PJ, Lai MY, Yang PM, Sheu JC, Wang TH, et al. Mixed infections of hepatitis C virus as a factor in acute exacerbations of chronic type C hepatitis. J Infect Dis 1994;170: 1128e33. [29] Wang Y, Okamoto H, Tsuda F, Nagayama R, Tao QM, Mishiro S. Prevalence, genotypes, and an isolate (HC-C2) of hepatitis C virus in Chinese patients with liver disease. J Med Virol 1993; 40:254e60. [30] Li JS, Tong SP, Vitvitski L, Lepot D, Trepo C. Evidence of two major genotypes of hepatitis C virus in France and close relatedness of the predominant one with the prototype virus. J Hepatol 1991;13(Suppl. 4):S33e7. [31] Hino K, Sainokami S, Shimoda K, Iino S, Wang Y, Okamoto H, et al. Genotypes and titers of hepatitis C virus for predicting response to interferon in patients with chronic hepatitis C. J Med Virol 1994;42:299e305. [32] Buckton AJ, Ngui SL, Arnold C, Boast K, Kovacs J, Klapper PE, et al. Multitypic hepatitis C virus infection identified by realtime nucleotide sequencing of minority genotypes. J Clin Microbiol 2006;44:2779e84. [33] Butt S, Idrees M, Ur Rehman I, Akbar H, Shahid M, Afzal S, et al. Mixed genotype infections with hepatitis C virus, Pakistan. Emerg Infect Dis 2011;17:1565e7. [34] Zarkesh-Esfahani SH, Kardi MT, Edalati M. Hepatitis C virus genotype frequency in Isfahan province of Iran: A descriptive cross-sectional study. Virol J 2010;7:69. [35] Viazov S, Widell A, Nordenfelt E. Mixed infection with two types of hepatitis C virus is probably a rare event. Infection 2000;28:21e5. [36] Toyoda H, Fukuda Y, Hayakawa T, Kumada T, Nakano S, Takamatsu J, et al. Presence of multiple genotype-specific antibodies in patients with persistent infection with hepatitis C virus (HCV) of a single genotype: evidence for transient or occult superinfection with HCV of different genotypes. Am J Gastroenterol 1999;94:2230e6. [37] Isobe K, Imoto M, Fukuda Y, Koyama Y, Nakano I, Hayakawa T, et al. Hepatitis C virus infection and genotypes in Japanese hemophiliacs. Liver 1995;15:131e4. [38] Schroter M, Feucht HH, Zollner B, Schafer P, Laufs R. Multiple infections with different HCV genotypes: prevalence and clinical impact. J Clin Virol 2003;27:200e4. [39] Hsu CS, Liu CH, Liu CJ, Wang CC, Chen CL, Lai MY, et al. Association of lipid profiles with hepatitis C viral load in chronic hepatitis C patients with genotype 1 or 2 infection. Am J Gastroenterol 2009;104:598e604. [40] Thompson AJ, Muir AJ, Sulkowski MS, Ge D, Fellay J, Shianna KV, et al. IL28B polymorphism improves viral kinetics and is the strongest pre-treatment predictor of SVR in HCV-1 patients. Gastroenterology 2010;139:120e9.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.kjms.2015.02.004.
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ORIGINAL ARTICLE
Host and virological characteristics of patients with hepatitis C virus mixed genotype 1 and 2 infection Chung-Feng Huang a,b, Ching-I Huang a,c, Ming-Lun Yeh c, Jee-Fu Huang c,d, Ming-Yen Hsieh c, Zu-Yau Lin c,d, Shinn-Cherng Chen c,d, Ming-Lung Yu c,d, Chia-Yen Dai c,d,e,f,*, Wan-Long Chuang c,d a
Graduate Institute of Clinical Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan b Department of Occupational Medicine, Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan c Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan d Faculty of Internal Medicine, School of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan e Department of Occupational Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan f Department of Preventive Medicine, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan Received 3 July 2014; accepted 6 February 2015
Available online 12 March 2015
KEYWORDS Genotype; Hepatitis C virus; Interleukin-28B; Mixed infection
Abstract The prevalence and characteristics of patients with hepatitis C virus mixed genotype 1 and 2 infection (HCV-1/2) remains unclear. For each HCV-1/2 patient with histological data available, two age- and sex-matched HCV-1 and HCV-2 infected patients were selected for comparison, respectively. Of the 2776 patients, 261 (9.4%) patients were identified as having mixed HCV-1/2 infection. The histological severity did not differ among HCV-1/2 patients and controls. The proportion of patients with the interleukin-28B (IL-28B) rs8099917 TT genotype did not differ between patients with mixed-1/2 and HCV-1 infection (82.6% vs. 86.5%, p Z 0.38). However, HCV-2 infected patients had a significantly higher proportion of the rs8099917 TT genotype compared to patients with mixed HCV-1/2 infections (91.6% vs. 82.6%, p Z 0.03). The HCV RNA levels were similar in patients with HCV-1/2 and HCV-1 infections (5.5 0.8 log IU/mL vs. 5.5 0.9 log IU/L, p Z 0.73), which were both significantly
Conflicts of interest: All authors declare no conflicts of interest. * Corresponding author. Hepatobiliary Division, Department of Internal Medicine, Kaohsiung Medical University Hospital, No. 100, Tzyou 1st Road, Kaohsiung 807, Taiwan. E-mail addresses: [email protected], [email protected] (C.-Y. Dai). http://dx.doi.org/10.1016/j.kjms.2015.02.004 1607-551X/Copyright ª 2015, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
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C.-F. Huang et al. higher than that of HCV-2 infection (5.1 0.9 log IU/mL, both p < 0.001). Linear regression analysis revealed that the factors of HCV-1/2 infection [b: 0.204; 95% confidence interval (CI): 0.183e0.598, p < 0.001], body weight (b: 0.150; CI: 0.003e0.021, p Z 0.008), and age (b: 0.139; CI: 0.020 to 0.002, p Z 0.01) were independently correlated with HCV RNA levels in patients with HCV-2 and mixed HCV-1/2 infection. We concluded that the host genetic and viral presentations of patients with mixed HCV-1/2 infection were more similar to those of patients with HCV-1 infection than patients with HCV-2 infection. Copyright ª 2015, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction Hepatitis C virus (HCV) infection is a major health issue that affects an estimated 170 million people worldwide [1,2]. An increased incidence of HCV-related end-stage liver disease and hepatocellular carcinoma has been noted in the past decade [3,4]. There are six HCV genotypes and more than 50 subtypes, which tend to segregate geographically. Regional distributions of HCV genotypes are diverse, reflecting the variability of acquisition routes and ethnic differences. HCV genotypes 1 (HCV-1), 2, and 3 have a fairly broad geographical distribution, whereas HCV genotypes 4, 5, and 6 are generally restricted to specific regions [5,6]. The understanding of viral genotype not only facilitates HCV treatment on an individualized basis [6e8] but also helps to elucidate the pathological nature of HCV infection [9]. For example, patients with HCV-1 infection have a lower sustained response (SVR) rate than patients with HCV-2. HCV-1b has been shown to have a higher risk for developing into hepatocellular carcinoma compared to other viral genotypes [9]. However, interleukin 28B (IL-28B) genetic variants have been recognized as the most important host factor either in the treatment of HCV infection [10e14] or in the influence of spontaneous HCV clearance [15]. We have demonstrated that viral genotype differences regarding IL-28B genotype distribution exist, which might in part be responsible for different treatment responses between HCV-1 and HCV-2 [16]. We have recently demonstrated that the treatment efficacy of patients with mixed HCV-1 and HCV-2 infection (HCV-1/2) was satisfactory in Asian patients receiving pegylated interferon/ribavirin combination therapy [17]. However, the prevalence of patients with mixed HCV-1/2 infection is unclear, and the clinical presentation, in terms of viral and host characteristics, such as IL-28B genetic variants, remains elusive. Taiwan possesses a unique epidemiological background because HCV is widely endemic and both HCV-1 and HCV-2 prevail throughout the entire country [6,18,19]. It is essential to fully clarify the host and virological characteristics of this population before the treatment strategy can be recommended. We herein retrospectively analyzed a hospital-based cohort with a respectable sample size to address this issue.
Materials and methods Eligible individuals were treatment-naı¨ve chronic hepatitis C patients that were enrolled consecutively in a medical
center and two core regional hospitals in southern Taiwan from 2001 to 2011. First, we analyzed the prevalence and characteristics of all the patients with mixed HCV-1/2 infection in the hospital cohort. To clarify the differences between mixed infections and other viral genotypes, patients with mixed HCV-1/2 infection with available histological data were selected to compare with age- and sexmatched HCV-1 and HCV-2 controls. All of the patients provided written informed consent prior to enrollment. Anti-HCV antibodies were detected using a thirdgeneration, commercially available enzyme-linked immunosorbent assay kit (AxSYM 3.0; Abbott Laboratories, Chicago, IL, USA). Serum HCV RNA was obtained using realtime polymerase chain reaction (COBAS AMPLICOR Hepatitis C Virus Test, ver. 2.0; Roche, Branchburg, NJ, USA; detection limit: 50 IU/mL). HCV genotypes were determined using the Okamoto method [20]. Liver histology was graded and staged according to the scoring system described by Knodell and Scheuer [21]. Biochemical items were measured on a multichannel auto analyzer (Hitachi Inc., Tokyo, Japan). The IL-28B genotypes of the patients were determined by the ABI TaqMan single nucleotide polymorphism (SNP) genotyping assays (Applied Biosystems, Foster City, CA, USA) using the predesigned commercial genotyping assays (ABI Assay ID: C__11710096_10). Briefly, polymerase chain reaction (PCR) primers and two allelespecific probes were designed to detect the specific SNP target. The PCR reactions were performed in 96-well microplates using ABI 7500 real-time PCR (Applied Biosystems). Allele discrimination was achieved by detecting fluorescence using ABI System SDS software version 1.2.3 (Applied Biosystems). The institutional review board at Kaohsiung Medical University Hospital approved the protocol, which conformed to the guidelines of the International Conference on Harmonization for Good Clinical Practice.
Statistical analyses IL-28B rs8099917 was selected as the candidate SNP analysis in the current study based on findings in our previous studies [10,11,22]. The frequency was compared between groups using the c2 test, with the Yates correction or Fisher’s exact test. Group means, presented as the mean values and standard deviation, were compared using analysis of variance and the Student t test or the ManneWhitney U test. Serum HCV RNA levels were expressed after logarithmic transformation of original values. To represent the severity of liver fibrosis, the aspartate
HCV mixed infection
273
aminotransferase (AST)-to-platelet ratio index (APRI) was calculated using the following equation: (AST level/upper limit of normal range)/platelet counts (109/L) 100 [23,24]. The frequencies of the rare allele of the rs8099917 genotype were too low; thus, the rare homozygote and heterozygote were combined together while analyzing the SNP. Logistic regression and linear regression analysis were performed to evaluate the independent factors associated with HCV RNA levels. The statistical analyses were performed using the SPSS 12.0 statistical package (SPSS, Chicago, IL, USA). All statistical analyses were based on twotailed hypothesis tests with a significance level of p < 0.05.
Results Overall patient characteristics in the hospitalbased cohort Of the 2776 patients, 311 (11.2%) were identified with HCV mixed infection and 261 (9.4%) were identified with mixed HCV-1/2 infections (Fig. 1). The basic demographic, virological, and clinical features of the 261 patients are shown in Table 1. The mean age was 51.0 11.9 years. Males accounted for 59.4% of the population. The mean HCV RNA levels were 5.7 0.9 log IU/mL. Among the 184 patients with available IL-28B genotype information, the rs8099917 TT genotype accounted for 154 (83.7%) patients, and 30 (16.3%) patients had the rs8099917 non-TT genotype. The majority of mixed HCV infections in patients were of the 1b/2a genotype (44.8%), followed by the 1b/2b genotype (43.7%). The clinical presentations between the two subpopulations were identical except that patients with HCV-1b/2b infection were younger and had higher HCV RNA levels (Table S2).
Characteristics of mixed HCV-1/2 infection in comparison with other HCV genotypes A total of 104 patients had histological data available. The characteristics did not differ between the patients with or without available histology data except that patients with
Table 1 Basic demographic and clinical features of the 261 patients with mixed HCV-1 and HCV-2 infection. Sex, n (%) Male Female Age (y), mean SD Body weight (kg), mean SD Rs8099917 TT genotype, n/N (%)a Baseline HCV RNA (log IU/mL), mean SD Baseline viral loads > 400,000 IU/mL, n (%) Fibrosis, n (%) 0e2 3e4 HAI, mean SD AST (IU/L), mean SD ALT (IU/L), mean SD GGT (IU/L), mean SD APRI, mean SD Fasting sugar (mg/dL), mean SD Genotype distribution, n (%) 1a þ 2a 1b þ 2a 1b þ 2b 1a þ 2a þ 2b 1a þ 1b þ 2a þ 2b 1a þ 1b þ 2a
155 (59.4) 106 (40.6) 51.0 11.9 67.4 12.9 154/184 (83.7) 5.7 0.9 157 (60.2) 76 (73.1) 28 (26.9) 5.1 2.7 94.4 68.0 144.3 154.8 76.4 78.5 2.6 2.1 102.1 34.8 2 (0.8) 117 (44.8) 114 (43.7) 26 (10.0) 1 (0.4) 1 (0.4)
ALT Z aspartate aminotransferase; APRI Z alanine aminotransferase to platelet ratio index; AST Z alanine aminotransferase; GGT Z gamma-glutamyl transferase; HAI Z histological activity index; HCV Z hepatitis C virus; SD Z standard deviation. a Data available in 184 patients.
unavailable data were older and had lower AST levels (Table S1). To fully explore the characteristics of patients with mixed HCV-1/2 infection, patients that were age- and sex-matched and infected with either HCV-1 or HCV-2 were selected for comparison with 1:2 ratios (Fig. 1). As shown in Table 2, the histological severity in terms of liver fibrosis and histological activity index did not differ among patients
2776 treatment-naïve CHC patients
Pure HCV-1 (n = 1294, 46.6%)
Pure HCV-2 (n = 1162, 41.9% )
Mixed HCV genotypes (n = 311, 11.2%)
Other genotypes (n = 9, 0.3%)
Mixed other genotypes (n = 50,1.8%)
208 age-, sex-matched HCV-1 and HCV-2 controls, respectively
Figure 1.
Histology available (n = 104)
Histology unavailable (n = 157)
Distribution of HCV genotypes in the cohort. CHC Z chronic hepatitis C; HCV Z hepatitis C virus.
274 Table 2
C.-F. Huang et al. Basic demographic and clinical features of the three patient groups with different HCV genotypes.
Sex, n (%) Male Female Age (y), mean SD Body weight (kg), mean SD Rs8099917 TT genotype, n/N (%) a Fibrosis, n (%) 0e2 3e4 HAI, mean SD Baseline HCV RNA (log IU/mL), mean SD AST (IU/L), mean SD ALT (IU/L), mean SD GGT (IU/L), mean SD APRI, mean SD Fasting sugar (mg/dL), mean SD
Mixed HCV-1/2 (n Z 104)
HCV-1 (n Z 208)
HCV-2 (n Z 208)
64 (61.5) 40 (38.5) 48.8 12.1 66.3 11.8 71/86 (82.6) *
125 (60.1) 83 (39.9) 48.5 11.4 65.8 10.2 180/208 (86.5)
128 (61.5) 80 (38.5) 47.9 10.8 65.5 11.3 174/190 (91.6) *
76 (73.1) 28 (26.9) 5.1 2.7 5.5 0.8 **
152 (73.4) 55 (26.6) 4.8 2.6 5.5 0.9 **
160 (77.7) 46 (22.3) 5.0 2.7 5.1 1.0 **
105.1 64.6 158.6 109.5 77.2 70.6 2.9 2.2 105.8 46.0
97.8 59.3 149.8 87.9 60.9 50.6 2.7 2.0 100.9 26.3
103.2 65.6 167.0 110.3 65.9 57.8 2.9 2.5 102.8 39.2
p 0.94
0.78 0.82 0.08 0.31
0.7 < 0.001 ** 0.54 0.23 0.07 0.51 0.54
* p Z 0.03; ** p < 0.001. ALT Z aspartate aminotransferase; APRI Z alanine aminotransferase to platelet ratio index; AST Z alanine aminotransferase; GGT Z gamma-glutamyl transferase; HAI Z histological activity index; HCV Z hepatitis C virus; mixed HCV-1/2 genotype Z HCV genotype 1 and HCV genotype 2 mixed infection; SD Z standard deviation. a Data available in 484 patients.
with mixed HCV-1/2, HCV-1, and HCV-2 infection. The proportion of the IL-28B rs8099917 TT genotype did not differ between patients with mixed-1/2 and HCV-1 infection (82.6% vs. 86.5%, p Z 0.38). However, HCV-2 infected patients had a significantly higher proportion of the rs8099917 TT genotype compared to patients with mixed HCV-1/2 infection (91.6% vs. 82.6%, p Z 0.03). The HCV RNA levels were similar between patients with HCV-1/2 and
Table 3
HCV-1 infection (5.5 0.8 log IU/mL vs. 5.5 0.9 log IU/L, p Z 0.73), which were both significantly higher than that of HCV-2 infection (5.1 0.9 log IU/mL, both p < 0.001). The HCV RNA levels were so similar between patients with mixed HCV-1/2 and HCV-1 infection that we further analyzed the characteristics associated with HCV RNA levels among patients with HCV-2 and mixed HCV-1/2 infection. As shown in Table 3, younger age, higher body
Factors associated with high or low viral loads among patients with HCV-2 and mixed HCV-1/2 infection. High viral loads (n Z 130)
Male sex, n (%) Age (y), mean SD Rs8099917 TT genotype, n/N (%) b Fibrosis 3/4, n/N (%) HAI, mean SD Body weight (kg), mean SD AST (IU/L), mean SD ALT (IU/L), mean SD GGT (IU/L), mean SD APRI, mean SD Fasting sugar (mg/dL), mean SD HCV genotype, n (%) HCV-2 Mixed HCV-1/2
Low viral loads (n Z 182)
86 (66.2) 46.5 12.8 99/115 (86.1) 31/130 (23.8) 5.1 2.8 68.1 11.4 100.0 64.8 165.4 119.4 72.1 59.8 2.6 2.2 103.7 31.8
106 (58.2) 49.5 9.9 146/161 (90.7) 43/180 (26.4) 4.8 2.6 64.1 11.1 106.6 65.5 163.3 103.0 67.8 64.4 3.1 2.5 103.9 47.6
73 (56.2) 57 (43.8)
135 (74.2) 47 (25.8)
p
0.16 0.03 0.23 0.99 0.12 0.002 0.38 0.87 0.55 0.06 0.96 0.001
a
Logistic regression analysis of factors associated with high viral loads OR
95% CI
p
0.979
0.958e1.000
1.030
1.008e1.052
0.007
2.171
1.324e3.559
0.002
0.05
ALT Z aspartate aminotransferase; APRI Z alanine aminotransferase to platelet ratio index; AST Z alanine aminotransferase; CI Z confidence interval; GGT Z gamma-glutamyl transferase; HAI Z histological activity index; HCV Z hepatitis C virus; mixed HCV-1/ 2 Z HCV genotype 1 and HCV genotype 2 mixed infection; OR Z odds ratio; SD Z standard deviation. a High viral loads defined as HCV RNA > 400,000 IU/Ml. b Data available in 276 patients.
HCV mixed infection
275
Table 4 Linear regression analysis of factors associated with viral loads among patients with HCV-2 and mixed HCV1/2 infection. a b Body 0.012 weight Mixed 0.391 HCV-1/2 genotype Age 0.011
Standard error
95% CI for b
b
p
0.004
0.003
0.021
0.150
0.105
0.183
0.598
0.204 < 0.001
0.004
0.020 0.002 0.139
0.008
0.01
CI Z confidence interval; HCV Z hepatitis C virus; mixed HCV1/2 genotype Z HCV genotype 1 and HCV genotype 2 mixed infection. a Odds ratios (ORs) are for body weight (per 1 kg increase), age (per 1 year increase), and HCV genotype (mixed HCV genotype 1and 2 vs. HCV genotype 2).
weight, and mixed HCV-1/2 infection were factors associated with high viral loads, defined as HCV RNA levels > 400,000 IU/mL, in univariate analysis. Multivariate analysis revealed that the strongest factor associated with high viral loads was mixed HCV-1/2 infection [odds ratio (OR)/95% confidence interval (CI): 2.171/1.324e3.559, p Z 0.002] followed by body weight (OR/CI: 1.030/1.008e1.052, p Z 0.007), and age (OR/CI: 0.979/0.958e1.000, p Z 0.05). Linear regression analysis revealed that the factors of HCV1/2 infection (b: 0.204; 95% CI: 0.183e0.598, p < 0.001), body weight (b: 0.150; 95% CI: 0.003e0.021, p Z 0.008) and age (b: 0.139; 95% CI: 0.020e0.002, p Z 0.01) were independently correlated with HCV RNA levels (Table 4).
The interplay of HCV RNA levels and host IL-28B genotype As shown in Table 5, the HCV RNA levels did not differ between HCV-1 patients with the rs8099917 TT genotype and the rs8099917 non-TT genotype (5.49 0.94 log IU/mL vs. 5.51 0.70 log IU/mL, p Z 0.92). The HCV RNA levels also did not differ between mixed HCV-1/2 patients with the rs8099917 TT genotype and the rs8099917 non-TT genotype (5.60 0.83 log IU/mL vs. 5.55 0.74 log IU/mL, p Z 0.92). However, the HCV RNA levels were significantly lower in HCV-2 patients with the rs8099917 TT genotype compared with those with the rs8099917 non-TT genotype (5.07 0.95 log IU/mL vs. 5.57 0.54 log IU/mL,
Table 5
p Z 0.003). The difference in HCV RNA levels between HCV2 and other HCV genotypes existed only in patients with the rs8099917 TT genotype (p < 0.001) but not in those with the rs8099917 non-TT genotype (Table 5). Among patients with the rs8099917 TT genotype, patients with mixed HCV-1/2 infection had a significantly higher proportion of high viral loads compared to patients with HCV-2 infection (59.2% vs. 33.9%, p < 0.001), and the proportion of high viral loads did not differ between non-TT genotype carriers with HCV-2 and mixed HCV-1/2 infection (53.3% vs. 50.0%, p Z 1) (Fig. 2).
Discussion To our knowledge, this is the first study to address the prevalence and characteristics of patients with mixed infections with HCV-1 and HCV-2. We demonstrated that one out of 10 HCV patients was infected with mixed HCV-1/2 in Taiwan. The liver disease severity, in terms of necroinflammation and fibrosis, was similar between the study population and those infected with other viral genotypes. Nevertheless, the host IL-28B genetic predisposition and viral presentation of patients with mixed HCV-1/2 infection were more similar to those with HCV-1 infection. Compared with patients with HCV-2 infection, patients with mixed HCV-1/2 infection had a significantly lower proportion of favorable IL-28B genotype carriage and higher HCV RNA levels. Superinfection of heterologous HCV in the same host was first demonstrated in chimpanzees [25,26]. Presentation as acute hepatitis in humans was reported shortly thereafter in early 1990 [27] and was further confirmed in a subsequent case-series study [28]. The definition of mixed HCV genotype infections is flexible and complex, as they might be comprised of different major genotypes or subtypes. The prevalence of mixed HCV genotype infections is also difficult to ascertain, with ranges from 0% to 25% having been reported depending on the genotyping method, definition of mixed infection, and study population [29e37]. The presence of mixed infections has been proposed as a potential cause for poor response to interferon-based antiviral therapy, as selection of minority genotype populations more refractory to antiviral treatment can occur during treatment [38]. The precision of studies on the transmission of HCV also depends on whether coexisting genotypes can be tracked along with the other ones. In the current study, we investigated patients with mixed infections of HCV-1 and HCV-2 for two reasons: first, the
HCV RNA levels in different HCV genotypes stratified by IL-28B genotype. Rs8099917 TT genotype
HCV-1 and mixed HCV-1/2 (log IU/mL), mean (SD) Mixed HCV-1/2 (log IU/mL), mean (SD) HCV-1 (log IU/mL), mean (SD) HCV-2 (log IU/mL), mean (SD)
5.50 5.60 5.46 4.99
(0.94) * (0.83) (0.97) * (1.02) *
Rs8099917 non-TT genotype 5.53 5.55 5.51 5.57
(0.70) (0.74) (0.70) (0.54)
p 0.87 0.84 0.80 0.003
* p < 0.001. HCV Z hepatitis C virus; IL-28B Z interleukin 28B; mixed HCV-1/2 Z HCV genotype 1 and HCV genotype 2 mixed infection; SD Z standard deviation.
276
Figure 2. Percentage of patients with high HCV RNA levels between patients with mixed HCV-1/2 and HCV-2 infection. The black bar represents patients with mixed HCV-1/2 infection. The white bar represents patients with HCV-2 infection. HCV Z hepatitis C virus; HVL Z high viral loads, defined as HCV RNA levels >400,000 IU/mL.
patients might be frequently encountered in daily practice in countries where both HCV genotypes prevail. Second, treatment strategies toward the two genotypes are largely different. It is therefore more rational and very essential to explore the characteristics of the virus and patients before we can fully address the treatment efficacy in this specific population. It has been suggested that patients infected with HCV-1 have higher HCV RNA levels compared with patients with HCV-2 infection [39]. Intriguingly, we demonstrated that patients with mixed HCV-1/2 infection had similar HCV RNA levels to patients with nonmixed HCV-1 but significantly higher HCV RNA levels than those with HCV-2 infection. Higher body weight was also independently associated with high viral loads. The precise reason was uncertain. Metabolic disarrangement in terms of insulin resistance and lipid disorders have been associated with HCV viral loads [39], which might be partially responsible for the result. Host IL-28B genetic variants are currently the most important host factor in determining viral eradication either by antiviral therapy [10e14] or self-limited HCV infection [15]. The distribution of IL-28B genetic variants would account for diverse treatment outcomes not only across different ethnicities of patients but also different viral genotypes [16]. We have previously demonstrated that the favorable IL-28B rs8099917 TT genotype was overrepresented among HCV-non-1 infected patients, compared with HCV-1 patients (91.4% vs. 85.0%, p Z 0.002) [16]. In the current study, we noticed that IL-28B genetic distribution in patients with mixed HCV-1/2 infection was more similar to patients with HCV-1 infection, with a lower proportion of favorable genotype carriage. Further studies to explore the treatment efficacy of the population based on these findings are warranted. Although patients were stratified according to IL-28B genotype, lower HCV RNA levels were only noted in HCV-2 patients with the favorable rs8099917 TT genotype. It has been suggested that HCV-1 patients with a favorable IL-28B genotype have higher HCV RNA levels [12], which might attribute to viral selection during evolution. However, the
C.-F. Huang et al. results were not consistent across all studies with different HCV genotypes [22,40]. The conflicting finding might in part result from selection bias of hospital-based studies. Further community-based studies enrolling larger sample size might be needed before a definite conclusion of the issue could be made. The limitation of the current study was that we failed to determine the predominant viral genotype in a given patient. To address this issue, a detailed cloning or deep sequence analysis might provide more precise evaluation. However, it is complicated and time-consuming and therefore is difficult to be applied to large-scale studies. Since the acquisition of HCV is frequently asymptomatic, we failed to judge the impact of duration of infection on liver fibrosis among study groups. The viral interference of heterologous HCV genotypes in the same host might be dynamic during the course of superinfection [18,28]. However, patients selected in the current hospital cohort were chronically infected and had been scheduled to receive antiviral therapy, so the viral presentation of mixed HCV1/2 infection in the same host was consistent in the current patient cohort. In conclusion, patients with mixed HCV-1/2 infection are frequently encountered in Taiwan. This should also be the case in areas where both viral genotypes are endemic. Both the viral and host genetic features in patients with mixed HCV-1/2 infection were more similar to those with HCV-1 infection than those with HCV-2 infection. In the era of direct antiviral agents (DAAs), so far, there is no solution for how to manage this specific population, unless pangenotypic DAAs are made widely available. The current study might provide insight and further guidance regarding the treatment of patients with mixed infections of HCV-1 and HCV-2.
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Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.kjms.2015.02.004.
