Chemokine levels were measured on a total of 9 subjects with acute HCV infection with a mean age of 23 years (). Of these, 6 were men and 3 were women, 8 were Caucasian. Eight study participants had primary acute HCV infections; one participant who had been infected previously, as indicated by HCV seropositivity at enrollment, had acute reinfection. One subject had two HCV infections with at least 6 months and 15 negative HCV RNA samples between the infections. Consequently, we were able to analyze chemokine levels in nine subjects with a total of ten HCV infections (eight primary infections and two reinfections).
Patient demographics and infection outcomes
In terms of the outcome, two participants resolved the infection (defined as at least six months of undetectable HCV RNA), one on two occasions. One participant cleared HCV following antiviral therapy, 4 developed chronic HCV infection, and 2 discontinued their participation in the study at weeks 7 and 17 after infection. Per patient, we measured CXCL10 on a mean of 28 (median 26) and CXCL9 and 11 on a mean of 27 (median 26) samples. On average, 23 quantitative HCV RNA measurements (median 18) and 25 ALT measurements (median 19) were available per patient. The average time between samples was 12 days for measurements of CXCR3-associated chemokines, 14 days for measurements of CCR5-associated chemokines, 13 days for ALT, and 10 days for HCV RNA measurements. Time between samples was calculated during the period from 50 days prior to 300 days after viral acquisition, the same time interval used for the statistical analysis
Changes in chemokine, ALT, and HCV RNA levels during acute HCV infection are illustrated in . While we observed increases in CXCR3-associated chemokines, neither CCL3 nor CCL4 changed appreciably following infection. Notably, CXCL9-11 induction did not occur early in infection, but was delayed for several weeks in all patients. Chemokine increases were followed by ALT elevation that demonstrated a similar pattern. Moreover, in the first 70 days post infection, we observed a strong similarity in the “sawtooth” patterns between chemokines, ALT and HCV RNA (most visually pronounced in subjects 00059, 00105, 00108). The fold increase for all three CXCR3-associated chemokines appears to be comparable. Both chemokines and ALT decrease 3–4 months post infection. However, among patients with chronic HCV infection, chemokine levels remain elevated compared to their initial values.
Individual plots illustrating HCV RNA, chemokine, and ALT values for the nine subjects included in the study
The reconstructed mean trajectory illustrates smooth increases in CXCR3-associated chemokines and ALT, peaking between 70 and 85 days (). These trajectories are estimated through non-parametric smoothing techniques and are based on 129 observations for HCV RNA, 166 for ALT, 195 for CXCL9, 200 for CXCL10 and 190 for CXCL11. After the chemokine peak, there is a gradual slow decrease that is more gradual than the increase initially observed. We also observe precipitous decrease in HCV RNA levels where quantitative measurements are available. As illustrated in , we noted a second peak in HCV RNA. However, while the over-all decline of HCV RNA is supported by the data, the second peak results from the sparse and non-uniform quantitative HCV RNA measurements and is likely to be spurious CCR5-associated chemokines are not apparently affected by the infection.
Mean HCV RNA, chemokine, and ALT smoothed trajectories before and after HCV acquisition
The certainty of the estimated reconstructed trajectory can be gleaned from the inspection of the 5 and 95 percentiles of the bootstrapped reconstructed trajectory for each chemokine at each time point (). Confidence intervals appear to provide reasonable coverage of the estimated trajectories for subjects both with dense as well as sparse data. The peaks in CXCL9-11 and ALT levels are well resolved for subjects with dense data. Confidence intervals are consistently wider for those with sparse data. As noted previously, no specific patterns are consistently observed for CCL3 and CCL4 reconstructions.
Bootstrapped confidence intervals for reconstructed trajectories for subjects with A) dense and B) sparse data
Time to initial chemokine elevation
We examined the time to first elevation in measured CXCL9-11 levels, which we defined as at least a four-fold increase in levels over the average of the pretreatment values. Participants 00110 and 00257 were excluded from this analysis due to the small number of chemokine measurements during the first few months of the infection. Reinfections in participants 00240 and 00257 were also excluded from this analysis. On average, elevation in CXCL10 levels was first detected 38 days after HCV acquisition (range, 20–61 days). Elevations in CXCL9 and CXCL11 were first detected 52 (range, 27–72) and 53 (range, 27–77) days after infection, respectively. No differences in time to elevation were observed comparing subjects that cleared the infection relative to those that became chronically infected.
Time to peak chemokine levels
The time to maximum ALT and CXCL9-11 levels occurred approximately three months post infection. ALT peaked 75 (95%CI 32–90) days, CXCL10 peaked 72 (95%CI 44–113) days, CXCL9 peaked 83 (95%CI 63–114) days, and CXCL11 peaked 77 (95%CI 63–133) days after the onset of HCV infection. No differences in time to peak elevation were observed comparing subjects that cleared the infection relative to those that became chronically infected.
Correlation between chemokines, HCV RNA, and ALT levels
To investigate the short-term relationship between the chemokines, ALT and HCV RNA, we analyzed the correlation between departures of these parameters from the estimated long-term trends for all study participants utilizing all samples on which chemokine levels were measured. We found a high level of correlation among the CXCR3-associated chemokines (ρ[CXCL9, CXCL10]=0.602, p<0.001; ρ[CXCL9, CXCL11]=0.706, p<0.001; ρ[CXCL10, CXCL11]=0.428, p<0.001). When evaluating the correlations between the chemokines and the virus, we noted the strongest correlation between HCV RNA and CXCL10 (ρ=0.528, p=0.004). Evaluating the relationships between chemokines and ALT, we noted the strongest correlation between ALT and CXCL9 (ρ=0.452, p=0.008). For all other correlations between chemokines, HCV RNA, and ALT, ρ was <0.35 or were not significant at the 0.05 level.
Lag correlations for chemokines, HCV RNA, and ALT
We next computed correlations between markers at the current time and at one- and two-week lags. ALT showed positive correlation with all CXCR3-related chemokines measured in the previous week (ρ[CXCL10, ALT]=0.386, p=0.006; ρ[CXCL9, ALT]=0.390, p=0.024; ρ[CXCL11, ALT]=0.405, p<0.001). These findings indicate that an increase in chemokine levels is associated with a subsequent increase in ALT levels. CXCR3-associated chemokines also exhibited negative auto-correlation at one week lags (ρ[CXCL9, CXCL9]=−0.281, p=0.050; ρ[CXCL11, CXCL11]=−0.519, p<0.001; ρ[CXCL11, CXCL9]=−0.266, p=0.034; ρ[CXCL9, CXCL11]=−0.390, p=0.014) suggesting a short-term oscillation. ALT was positively auto-correlated at week 1 (ρ= 0.332, p=0.04) suggesting a possible oscillation at longer intervals. We also noted weak negative correlations between HCV RNA and CXCL9/CXCL11 measured two weeks before (ρ[CXCL9, HCV RNA]=−0.300, p=0.018; ρ[CXCL11, HCV RNA]=−0.267, p=0.048) while weak positive correlations were found between CXCL9 and two weeks following, HCV RNA (ρ[HCV RNA, CXCL9]=0.298, p<0.042). Correlations at larger lags beyond two weeks tended to be small.
In depth analysis of one participant with frequent sampling
Subject 00105 provided a particularly long and consistent pattern of weekly samples up to day 540 after HCV acquisition that revealed a qualitative change in the measurements at approximately day 210 (). At this point, HCV RNA levels increased substantially accompanied by a four-fold reduction in the volatility of measurements about the estimated long term trend (p<0.0001). A similar reduction in volatility was observed in ALT (p=0.0039) and in the CXCR3-associated chemokines (CXCL10: p<0.0001, CXCL9: p=0.0002, CXCL11: p=0.0077). After accounting for the change in volatility, similar patterns of Spearman’s rank correlation and lagged correlation were observed on both sides of the change point (). Correlations prior to the change point agreed well with the correlations observed in the pooled sample data for all study participants as reported above. No non-lagged correlation changed by more than a factor of 2. After the change-point, a consistent decrease was found in the one-week lag between ALT and the CXCR3-associated chemokines ρ[CXCL10, ALT]=0.10 to −0.23, ρ[CXCL9, ALT]=0.16 to −0.15, ρ[CXCL11, ALT]=0.21 to −0.06. The interpretation of correlations of repeated short-term oscillations in chemokines is visually apparent in in which HCV RNA and CXCR3-associated chemokines exhibit tightly-associated “sawtooth” patterns on a 2 to 3 week cycle while similar cycles are somewhat smoother in ALT.
Detailed evaluation of subject 00105