Abstract
Background and Objectives
Hepatitis C virus (HCV) RNA level in acute HCV infection is predictive of spontaneous clearance. This study assessed factors associated with HCV RNA levels during early acute infection among people who inject drugs with well-defined acute HCV infection.
Study design
Data were from International Collaboration of Incident HIV and Hepatitis C in Injecting Cohorts (InC3) Study, an international collaboration of nine prospective cohorts studying acute HCV infection. Individuals with available HCV RNA levels during early acute infection (first two months following infection) were included. The distribution of HCV RNA levels during early acute infection were compared by selected host and virological factors.
Results
A total of 195 individuals were included. Median HCV RNA levels were significantly higher among individuals with interferon lambda 3 (IFNL3, formerly called IL28B) CC genotype compared to those with TT/CT genotype (6.28 vs. 5.39 log IU/mL, respectively; P=0.01). IFNL3 CC genotype was also associated with top tertile HCV RNA levels (≥6.3 IU/mL; vs. TT/CT genotype; adjusted Odds Ratio: 4.28; 95%CI: 2.01, 9.10; P<0.01).
Conclusions
This study indicates that IFNL3 CC genotype predicts higher HCV RNA levels in early acute HCV infection.
Keywords: Viral load, acute HCV, IFNL3 genotype, IL28B genotype, Cohort study
Introduction
The dynamics of hepatitis C virus (HCV) RNA levels in acute HCV infection have been characterised as occurring in three phases: a pre-ramp-up phase with intermittent low-level HCV RNA (from exposure to initial quantifiable HCV RNA); a ramp-up phase with exponential increase in HCV RNA levels; and a high-titre viremic plateau phase1. Then, acute HCV infection is followed by spontaneous clearance in around 25% of individuals2, 3 while the remaining 75% progress to chronic HCV infection.
Higher HCV RNA levels during the first month of acute infection have been shown to be associated with spontaneous clearance4. However, there are limited studies investigating factors associated with HCV RNA levels during acute infection4, 5, and none evaluating multiple relevant factors simultaneously. A better understanding of factors associated with HCV RNA levels in early acute infection has the potential to assist in therapeutic decision making during acute HCV and also enhance our understanding of HCV immunopathogenesis and biological mechanisms for defining protective immunity, which is important for vaccine design.
This current study assessed factors associated with HCV RNA levels in early acute infection (first two months following infection) among people who inject drugs (PWID) in a large population with well-defined acute HCV infection.
Study Design
Study population
The International Collaboration of Incident HIV and Hepatitis C in Injecting Cohorts (InC3) Study, is a collaboration of pooled data from nine prospective international cohorts prominently following PWID, consisting of a large number of well characterized participants with acute HCV infection and longitudinal follow-up6. All cohorts follow participants at regular intervals using standardized methods.
Documented acute HCV is defined as either: 1) HCV seroconversion with an HCV antibody (anti-HCV) or HCV RNA positive test within two years of the anti-HCV negative test; or 2) evidence of symptomatic HCV infection (defined by a positive anti-HCV/HCV RNA test, jaundice or alanine transaminase (ALT) elevation >400 IU/L, and detection of HCV RNA or history of high-risk exposure within three months of clinical manifestation of acute HCV).
HCV RNA levels in early acute infection were assessed among InC3 participants with available HCV RNA tests during the first two months following infection. This period was chosen on the basis of our initial analysis7, 8 and also the other data4 demonstrating that this is the period where peak HCV RNA levels are observed during acute infection. From the total InC3 participants with acute HCV infection (n=812), individuals with unavailable HCV RNA tests during the first two months following infection were excluded (n=603). Individuals with undetectable HCV RNA during the first two months with repeated undetectable tests during follow-up were defined as having early spontaneous clearance and also excluded (n=14). As such, 195 individuals with available HCV RNA during the first two months following infection were included in this analysis.
The estimated date of HCV infection was calculated based on a hierarchy using all serological (anti-HCV), virological (HCV RNA) and clinical (symptoms and liver function tests) data to arrive at the most precise estimate of infection date:
Among individuals with HCV RNA positive and anti-HCV negative at acute HCV detection, date of infection was four weeks prior to HCV RNA detection9, 10.
Among individuals with symptomatic acute HCV, date of infection was six weeks prior to its onset (jaundice or ALT >400 IU/L)11.
Among individuals with a negative anti-HCV test followed by either a positive anti- HCV or HCV RNA test, seroconversion was assumed to occur at the mid-point between the last negative and the first positive test. HCV seroconversion generally occurs about 30-60 days following infection9, 10, 12. Date of infection in this group was six weeks prior to estimated seroconversion date if the first positive test was anti- HCV test and four weeks prior to estimated seroconversion date if the first positive test was only HCV RNA test.
Laboratory testing
Choice of qualitative and quantitative HCV RNA testing varied by cohort but was consistent at each site. Qualitative HCV RNA testing was performed using the following assays: Versant TMA [Bayer, Australia; <10 IU/ml], COBAS AmpliPrep/COBAS TaqMan (Roche, Branchburg, NJ, USA; <15 IU/ml), COBAS AMPLICOR HCV Test v2.0 (Roche Diagnostics, Mannheim, Germany; <50 IU/ml) or discriminatory HCV transcription-mediated amplification component of the Procleix HIV-1/HCV (Gen-Probe, San Diego, CA, USA; <12 copies/mL). Quantitative HCV RNA testing was performed using the Versant HCV RNA 3.0 (Bayer, Australia; <615 IU/ml), COBAS AMPLICOR HCV MONITOR 2.0 (Roche Diagnostics, Mannheim, Germany; <600 IU/ml), COBAS AmpliPrep/COBAS TaqMan (Roche, Branchburg, NJ, USA; <15 IU/ml) or an in-house PCR (<1000 IU/ml)13, 14. HCV genotype was determined by line-probe assay (Versant LiPa1/LiPa2, Bayer, Australia) or HCV sequencing at acute HCV detection. Among those with undetectable HCV RNA (no genotype) and available samples, Murex HCV serotyping was performed to determine HCV genotype (Murex Biotech Limited, Dartford, UK). Interferon lambda 3 (IFNL3) genotyping (formerly called interleukin 28 B [IL-28B]) was determined by sequencing of the rs12979860 single nucleotide polymorphism, as previously described in2, 15-17.
Study outcomes and statistical analyses
The study outcome was HCV RNA levels during the first two months following infection (early acute infection). Nonparametric statistical tests were used for analyses, given that HCV RNA levels (IU/mL) and log10 transformation of HCV RNA levels (log IU/mL) were not normally distributed. The top tertile HCV RNA levels (≥6.3 log IU/mL) was defined as a high HCV RNA level.
Previous data indicated that spontaneous clearance is associated with higher HCV RNA levels in early acute infection4. Therefore, factors hypothesized to be associated with HCV RNA levels were determined a priori based on the factors shown to be associated with spontaneous clearance, including age18, sex2, 3, 17, 19, ethnicity20, IFNL3 genotype (SNP rs12979860; CC vs. CT/TT)2, 15, 16, 21, HIV co-infection20, and HCV genotype2, 22.
Median HCV RNA levels were compared between groups using the Wilcoxon-Mann-Whitney (or Kruskal Wallis) test. Logistic regression models were also used to assess factors associated with high HCV RNA levels (≥6.3 log IU/mL). In multivariate regression analysis, initial models were adjusted for sex, IFNL3 genotype, and HCV genotype a priori given our data showed independent association between these variables and spontaneous clearance2. To account for potential unmeasured confounders introduced by cohort sites, multivariate regression analysis was performed using mixed modelling, with a random intercept for cohort site. Statistically significant differences were assessed at P<0.05 (P-values are two-sided). All analyses were performed using Stata v12.0 (College Station, TX, United States).
Results
One hundred and ninety-five individuals were included in the analysis. The median age was 24 years, 36% were female, 79% were Caucasian, and 3% were HIV co-infected (Table 1). Among those with data on infecting HCV genotype (n=172; 88%), 59% had genotype 1. Among those with data on IFNL3 genotype (n=161; 83%), 48% were IFNL3 CC genotype. One hundred and fifty individuals (77%) were anti-HCV negative/HCV RNA positive at acute HCV detection.
Table 1. Characteristics of participants with available HCV RNA levels during during the first two months following infection in the InC3 Study.
| Number (%) Total n=195 |
|
|---|---|
| Site | |
| ACS (the Netherlands) | 21 (11) |
| ATAHC (Australia) | 5 (3) |
| BAHSTION (United States) | 9 (5) |
| BBAASH (United States) | 72 (37) |
| HEPCO (Canada) | 4 (2) |
| HITS-c (Australia) | 2 (1) |
| HITS-p (Australia) | 23 (12) |
| N2 (Australia) | 0 (0) |
| UFO (United States) | 59 (30) |
|
| |
| Median age at the time of HCV infection, years (IQR) | 24 (21, 28) |
|
| |
| Sex | |
| Female | 64 (36) |
| Male | 122 (63) |
| Unknown | 1 (1) |
|
| |
| Ethnicity | |
| Caucasian | 155 (79) |
| Black | 12 (6) |
| Indigenous | 7 (4) |
| Other | 17 (9) |
| Unknown | 4 (2) |
|
| |
| History of injecting drug use | 195 (100) |
|
| |
| Symptomatic HCV infection | |
| No | 9 (5) |
| Yes | 15 (8) |
| Unknown | 171 (88) |
|
| |
| IFNL3 genotype (rs12979860) | |
| TT | 16 (8) |
| CT | 67 (34) |
| CC | 78 (40) |
| Unknown | 34 (17) |
|
| |
| HIV infection at the time of HCV infection | |
| No | 182 (93) |
| Yes | 6 (3) |
| Unknown | 7 (4) |
|
| |
| HCV genotype | |
| Genotype 1 | 102 (52) |
| Genotype 2 | 15 (8) |
| Genotype 3 | 49 (25) |
| Genotype 4 | 2 (1) |
| Mixed genotype | 4 (2) |
| Unknown | 23 (12) |
ACS: Amsterdam Cohort Studies; ATAHC: Australian Trial in Acute Hepatitis C; BAHSTION: Boston Acute HCV Study: Transmission, Immunity and Outcomes Network; BBAASH: Baltimore Before and After Acute Study of Hepatitis; HEPCO: St. Luc Cohort, HEPCO; HITS-c: Hepatitis C Incidence and Transmission Study-Community; HITS-p: Hepatitis C Incidence and Transmission Study-Prison; N2: Networks 2; UFO: UFO STUDY; IQR: Inter-quartile range
Median HCV RNA levels during early acute infection was 5.64 log IU/mL (Inter-quartile range [IQR]: 3.97, 6.70). Significantly higher median HCV RNA levels were observed among individuals with IFNL3 CC genotype compared to those with TT/CT genotype (6.28 vs. 5.39 log IU/mL, respectively; P=0.01). There was no significant difference in median HCV RNA levels by age, sex, ethnicity, HIV co-infection and HCV genotype (Table 2 and Figure 1).
Table 2. Median HCV RNA levels during the first two months following infection by selected demographic and virologic variables in participants with acute HCV infection in the InC3 study.
| Number Total n=195 |
Median HCV RNA levels* (IQR) | P | |
|---|---|---|---|
| Age | 0.31 | ||
| <30 years | 135 | 5.65 (4.32, 6.76) | |
| 30-39 years | 23 | 4.55 (1.70, 6.75) | |
| ≥40 years | 8 | 5.68 (5.07, 6.20) | |
|
| |||
| Sex | 0.69 | ||
| Female | 72 | 5.52 (3.93, 6.70) | |
| Male | 122 | 5.67 (4.07, 6.66) | |
|
| |||
| Ethnicity | 0.38 | ||
| Caucasian | 155 | 5.72 (3.97, 6.75) | |
| Black | 12 | 5.54 (5.10, 6.17) | |
| Indigenous | 7 | 5.74 (4.43, 5.83) | |
| Other | 17 | 4.81 (2.69, 6.04) | |
|
| |||
| IFNL3 genotype | 0.01 | ||
| TT/CT | 83 | 5.39 (4.46, 6.02) | |
| CC | 78 | 6.28 (3.45, 7.28) | |
|
| |||
| HIV status | 0.85 | ||
| Negative | 182 | 5.59 (3.97, 6.65) | |
| Positive | 6 | 5.14 (3.17, 6.75) | |
|
| |||
| HCV genotype | 0.50 | ||
| Genotype 1 | 102 | 5.67 (4.74, 6.70) | |
| Genotype 2 | 15 | 6.46 (3.82, 7.33) | |
| Genotype 3 | 49 | 5.36 (4.45, 6.39) | |
| Other† | 6 | 5.68 (1.70, 5.93) | |
IQR: Inter-quartile range
log IU/mL
Included genotype 4 (n=2), and mixed genotype (n=4).
Fig. 1.
Distribution of HCV RNA levels during the first two months following infection in participants with acute HCV infection in the InC3 study, stratified by (A) IFNL3 genotype, (B) sex, and (C) HCV genotype. Horizontal lines represent the medians HCV RNA levels in each subgroup.
In unadjusted logistic regression analysis (Table 3), IFNL3 CC genotype was the only factor significantly associated with high HCV RNA levels (≥6.3 log IU/mL). In a logistic regression model adjusting for sex, IFNL3 genotype, HCV genotype, and the cohort sites (Table 3), IFNL3 CC genotype remained independently associated with high HCV RNA levels (adjusted odds ratio: 4.28; 95%CI: 2.01, 9.10). Interactions between covariates were not statistically significant on the multiplicative scale.
Table 3. Logistic regression models assessing factors associated with HCV RNA levels ≥6.3 IU mL (top tertile) during the first two months following infection in participants with acute HCV infection in the InC3 study.
| HCV RNA levels ≥6.3 IU/mL n (%) | Unadjusted model | Adjusted model* | ||||
|---|---|---|---|---|---|---|
|
| ||||||
| OR (95% CI) | P | P overall | AOR (95% CI). | P | ||
| Age | 0.75 | |||||
| <30 years | 48 (36) | 1.00 | ||||
| 30-39 years | 7 (30) | 0.79 (0.30, 2.06) | 0.63 | |||
| ≥40 years | 2 (25) | 0.60 (0.12, 3.11) | 0.55 | |||
|
| ||||||
| Sex | ||||||
| Female | 23 (32) | 1.00 | 1.00 | |||
| Male | 42 (34) | 1.12 (0.60, 2.08) | 0.72 | 1.15 (0.53, 2.47) | 0.72 | |
|
| ||||||
| Ethnicity | 0.58 | |||||
| Indigenous | 1 (14) | 1.00 | ||||
| Caucasian | 59 (38) | 3.69 (0.43, 31.39) | 0.23 | |||
| Black | 2 (17) | 1.20 (0.09, 16.23) | 0.89 | |||
| Other | 3 (18) | 1.28 (0.11, 15.00) | 0.84 | |||
|
| ||||||
| IFNL3 genotype | ||||||
| TT/CT | 17 (20) | 1.00 | 1.00 | |||
| CC | 39 (50) | 3.88 (1.94. 7.77) | <0.01 | 4.28 (2.01, 9.10) | <0.01 | |
|
| ||||||
| HIV status | ||||||
| Negative | 58 (32) | 1.00 | ||||
| Positive | 3 (50) | 2.14 (0.42, 10.92) | 0.36 | |||
|
| ||||||
| HCV genotype | 0.26 | |||||
| Genotype 3 | 14 (29) | 1.00 | 1.00 | |||
| Genotype 1 | 35 (34) | 1.30 (0.62, 2.74) | 0.48 | 1.20 (0.51, 2.81) | 0.69 | |
| Genotype 2 | 8 (53) | 2.86 (0.87, 9.38) | 0.08 | 2.15 (0.49, 9.36) | 0.31 | |
| Other | 1 (17) | 0.50 (0.05, 4.67) | 0.54 | 0.46 (0.04, 5.05) | 0.52 | |
OR: Odds Ratio; CI: Confidence Interval; AOR: Adjusted Odds Ratio
Includes 149 participants in the model. The model was adjusted for site using a random intercept model
Discussion
In this current study, IFNL3 CC genotype was associated with higher HCV RNA levels in early acute infection, which is consistent with previous data4, 5. The strength of the current study was that we controlled for a range of factors potentially associated with spontaneous clearance, and the association between IFNL3 CC genotype and high HCV RNA levels (≥6.3 log IU/mL) remained strong even after adjustment. Previous studies have also demonstrated that IFNL3 CC genotype is associated with higher HCV RNA levels in chronic HCV infection23-25. Genetic variation in the IFNL3 gene region is a major host factor associated with both spontaneous and treatment-induced HCV clearance (reviewed in26). The exact mechanism underlying this genetic association remains to be determined. In one study of acute HCV infection, IFNL3 CC genotype was associated with higher initial HCV RNA levels, which was correspondingly associated with a greater likelihood of spontaneous clearance4. The authors suggested that high-level HCV replication could trigger stronger innate immune responses, thereby activating a stronger adaptive immune response that enhanced eradication of the virus4. Further research is needed to better elucidate the mechanisms behind the relationship between IFNL3 genotype, early HCV RNA levels and spontaneous clearance.
While the current study is unique given the large sample size and well-defined nature of early acute HCV infection, there are some limitations. Nine cohorts of individuals with acute HCV were combined. Participating cohorts bring a range of data types and structures presenting issues surrounding both inconsistent measurement and biological data testing protocols (e.g. HCV RNA assays differed across cohorts with different sensitivity, specificity and lower limit of detection). There were also small numbers for some categorized variables in this study (ethnicity and HIV status), limiting the statistical power of detecting associations between these variables and HCV RNA levels.
In conclusion, the current study identified that IFNL3 genotype predicts higher HCV RNA levels during the first two months following infection. These data provide better understanding of HCV immunopathogenesis during early acute infection. Further research is needed to understand the mechanism of IFNL3 genotype on HCV replication.
Highlights.
Factors associated with HCV RNA levels in early acute infection were evaluated.
Interferon lambda 3 (IFNL3) genotype was associated with HCV RNA levels.
Median HCV RNA levels were higher among those with IFNL3 CC genotype (vs. TT/CT).
IFNL3 CC genotype was associated with HCV RNA levels ≥6.3 IU/mL.
IFNL3 CC genotype predicts higher HCV RNA levels in early acute HCV infection.
Acknowledgments
Funding: The InC3 Study is supported by the National Institute on Drug Abuse Award Number R01DA031056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute on Drug Abuse or the National Institutes of Health. The Kirby Institute is funded by the Australian Government Department of Health and Ageing. The views expressed in this publication do not necessarily represent the position of the Australian Government.
Footnotes
Competing interests: The authors do not have any commercial or other association that might pose a conflict of interest.
Ethical approval: All participants provided written informed consent and cohort protocols were approved by local ethics committees.
Author's contributions: All authors contributed to the design of the InC3 Study. All authors have contributed data to the InC3 Study. BH drafted the study concept sheet, which was reviewed by JG, GJD, BG, AK, BM, KP, RBG and MP. The statistical analysis was conducted by BH, BG, and JG. JA reviewed the data analysis. BH, JG and GJD compiled the first draft of the manuscript, which was reviewed by all other authors. JG and GJD provided supervision of research. All authors contributed to and have approved the final manuscript.
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