Liver stiffness is not associated with short- and long-term plasma HIV RNA replication in immunocompetent patients with HIV infection and with HIV/HCV coinfection

Introduction

Chronic liver disease is a major cause of morbidity and mortality in human immunodeficiency virus (HIV)-positive patients, and coinfection with hepatitis C virus (HCV) is one of the most clinically important comorbidities [1]. However, many other factors, such as alcohol abuse, drug-related hepatotoxicity and nonalcoholic fatty liver disease, may also be responsible for liver damage [2,3]. This scenario is evolving as a result of the availability of direct-acting antivirals for the treatment of HCV infection and the current minimal exposure to antiretroviral drugs with a potentially hepatotoxic profile, such as didanosine, stavudine or zidovudine [4-6]. HIV may be directly responsible for liver damage; Kupffer cells, hepatocytes, and human stellate cells (HSCs) do not express CD4 receptors, and HIV cannot infect these cells, but the virus can stimulate HSCs through a C-C chemokine receptor type 5- or C-X-C chemokine receptor type 4-dependent pathway. HSCs are key cells involved in the progression of fibrosis: transforming growth factor-β, secreted by human peripheral blood mononuclear cells infected by HIV-1, was found to promote HSC modulation in an in vitro model [7,8]. Updated guidelines recommend that antiretroviral treatment (ART) should be started in all HIV-infected subjects with detectable plasma HIV viremia, regardless of CD4+ cell count; successful plasma HIV viremia suppression is achieved in most patients, but long-term efficacy may be suboptimal [6,9].

Liver fibrosis in HIV-positive patients is a multifactorial process; parameters modifiable over time (e.g., HBV-DNA and HCV-RNA detectability or undetectability) or parameters not modifiable over time (e.g., sex) may influence liver fibrosis [3,10,11]. The measurement of liver stiffness (LS) by transient elastography (TE) represents a rapid and noninvasive method for predicting liver fibrosis, and the result, which is expressed in kilopascals (kPa), is available immediately; this imaging modality is highly accurate in the detection of moderate fibrosis and cirrhosis in patients with HIV/HCV coinfection [12]. Abnormal LS values were found in a percentage of HIV-monoinfected patients, ranging from 41.9% (LS values >5.3 kPa were considered abnormal) to 11.2% (LS values ≥7.2 kPa were considered abnormal) [13-15].

TE was shown to be more reliable than the aspartate aminotransferase (AST)-to-platelet ratio index and the Fibrosis-4 score (FIB-4) in the staging of liver fibrosis in HIV/HCV coinfected patients; both indices include platelet count, which may be lower in viremic HIV-positive patients because of HIV-related thrombocytopenia [16,17]. The first aim of this study was to describe the demographic and viro-immunological parameters that influence TE results in a cohort of immunocompetent HIV-monoinfected patients and HIV/HCV-coinfected patients; the second aim of this study was to evaluate the reliability of a model (linear predictor) that included clinical variables in predicting LS value.

Patients and methods

We retrospectively enrolled patients into this study based on the following eligibility criteria: 1) age over 18 years; 2) diagnosis of chronic HIV infection; 3) hepatitis B serum antigen (HBsAg)-negative; 4) HCV treatment-naïve or previously treated relapsers or non-responders; 5) regular viro-immunological follow up of the HIV disease during at least the 12 months before TE; 6) no acute HCV infection; 7) no alcohol abuse; 8) no didanosine, stavudine or zidovudine therapy; and 9) valid TE performed between December 1, 2013, and September 30, 2015. Patients were excluded from the study if they showed a sustained virological response (SVR), experienced a spontaneous clearance of HCV, or had other known causes of liver disease or previous liver transplantation. Alcohol abuse was defined as the daily consumption of alcohol in doses of more than 20 g/day for women and more than 30 g/day for men. This study was conducted in accordance with local legislation and received approval from the local Ethics Committee of Padova University Hospital (61946/15). Anonymous data were collected retrospectively.

Patients were either untreated for HIV infection or received ART according to the current international guidelines. Decisions on HIV therapy were made at the discretion of the treating physician; there were no restrictions on CD4+ cell count.

Patients were classified based on plasma HIV viremia (detectable or undetectable when TE was performed and in the 12 months and the 36 months before TE) and on the presence of HCV infection (patients were either HCV-positive or HCV-negative). We defined T0 as the study time corresponding to the execution of TE, T1 as the study time corresponding to the 12-month period (short term) before TE and T2 as the study time corresponding to the 36-month period (long term) before TE. Plasma HIV viremia data during T2 were available for a subgroup of subjects.

Results A total of 413 patients underwent a TE; a complete description of the selection process is summarized in Fig. 1. Overall, 251 of the 277 (90.6%) patients were suitable for the main analysis (T1 study time); 26 patients were not included in the study because of a discordant plasma HIV viremia result at T1 and at T0 (21 patients had successful virological suppression at T0 and detectable viremia 12 months before TE).

Figure 1 Flow chart describing the selection of the study patients

1Alcohol abuse in 5 patients.

2Therapy with didanosine, stavudine or zidovudine in 3 patients TE, transient elastography.

The group of 251 patients included 178 HIV-monoinfected patients (124 with undetectable HIV RNA and 54 with detectable HIV RNA) and 73 HIV/HCV coinfected subjects (54 with undetectable HIV RNA and 19 with detectable HIV RNA). The demographic and viro-immunological characteristics of all the patients included in the study are presented in Table 1. Patients with HIV/HCV coinfection had a significantly higher LS value (P<0.0001), a greater incidence of significant fibrosis (P<0.0001), higher AST and alanine aminotransferase values (P<0.0001), and a lower number of CD4+ cell counts at nadir (P=0.003).

Table 1 Characteristics of the 178 HIV-monoinfected subjects and of the 73 HIV HCV coinfected patients

The 36-month study period was available for 154 subjects (61.3%), including 41 subjects (26.6%) with HIV/HCV coinfection and 113 subjects (73.4%) with HIV monoinfection. Most patients were Caucasian (96.8%) and on ART (89.6%). The HCV genotype was available for 72 of the 73 patients with a 12-month study duration; 41 of these subjects (56.9%) had genotype 1 infection. The mean HCV RNA level was comparable in coinfected patients with detectable pHIV and with undetectable pHIV (1,021,736 IU/mL, 95% confidence interval [CI] 393,401-2,653,638 IU/mL and 1,216,888 IU/mL, 95%CI 797,099-1,857,756 IU/mL, respectively; P=0.69). Only a few HIV/HCV patients (14 of 73, 19.2%) had been previously treated with an interferon-based regimen. The mean LS was higher in HIV/HCV-coinfected patients (P<0.0001); most HIV-monoinfected patients (91%) had an LS value ≤7.1 kPa. No significant differences in LS values were found between patients classified as having detectable or undetectable plasma HIV viremia, while HIV/HCV patients had higher LS values than monoinfected subjects, regardless of plasma HIV viremia (Fig. 2). There was a significant correlation between lkpa and age (P=0.025), HCV coinfection (P<0.0001) and AST (P<0.0001). The quadratic variable “bmi2” was added to the linear regression model in order to investigate the hypothesis of a non-linear correlation between lkpa and body mass index (BMI) and this hypothesis was confirmed (P=0.026); the other variables failed to reach significant P-values, including the successful suppression of HIV viremia at T1 and T2. This last finding was confirmed using logistic regression; this statistical approach revealed a positive correlation between an LS value >7.1 kPa and BMI (P=0.006), BMI as a quadratic variable (P=0.003), HCV coinfection (P<0.0001 and AST (P=0.001). The final linear regression model data are reported in Table 2; the mean predicted values were 4.69 kPa (95%CI 4.44-4.95 kPa) in patients with HIV monoinfection and 8.39 kPa (95%CI 7.70-9.15 kPa) in subjects with HIV/HCV coinfection. Fig. 3 depicts the effects of HCV infection and BMI on the original LS data and on the model-based predictions.

Figure 2 Liver stiffness values in HIV-monoinfected patients and in HIV/HCV patients according to plasma HIV RNA detectability or undetectability in the 12 months or 36 months before transient elastography

LS, liver stiffness; kPa, kilopascal; pHIV, plasma HIV RNA; m, months.

Table 2 Linear regression model with lkpa (liver stiffness absolute value after log₁₀ transformation), as dependent variable

Figure 3 Linear model representation including the three main variables, log₁₀(kPa), BMI, and HCV. The Figure depicts the effects of HCV infection and BMI on the Fibroscan values on the original data and on the model-based predictions

BMI, body mass index; CI, confidence interval; L. Fit, linear fit; Lowess, locally weighted regression; Obs, observations; HCV0, HIV patient HCV-negative; HCV 1, HIV patient HCV-positive.

Following the linear regression model, an analysis was performed to describe bivariate normal distributions for predicted and observed LS values in patients with and without HCV coinfection. Two subgroups of outliers were identified in the group with HIV monoinfection (8 patients in each subgroup, 9% of HIV-monoinfected patients) (Fig. 4). The predicted LS values included in the left outlier group were higher than or comparable to the observed LS values; conversely, the predicted LS data in the right outlier group were lower than the observed LS values. Patients included in the right outlier cohort had more frequent undetectable plasma HIV RNA (62.5% versus 12.5%, P=0.03) and lower mean AST (33 U/l versus 89 U/l, 95%CI of the difference 40-81 U/l, P<0.0001. Notably, the mean BMI was greater than 25 in both left outlier (28.6) and right outlier (29.6) groups (95%CI for combined groups 26.7-31.4).

Figure 4 Graph describing the linear fit between predictions and observed Fibroscan values.

The reference line where predictions correspond to observations is also shown. The ellipses indicate bivariate normal distributions for predictions and observed Fibroscan values. Each of them correspond to a probability of 86%; they account for the two subpopulations according to HCV status. Outliers, left: green; outliers, right, magenta;

Obs, observations; HCV 0, HIV patient HCV-negative; HCV 1, HIV patient HCV-positive; Out, outliers.

Discussion

We performed a linear model analysis to describe the correlations between clinical and viro-immunological variables and LS measurement (LSM) in a cohort of patients with HIV infection or with HIV/HCV coinfection, focusing on the role of plasma HIV viremia detectability or undetectability during two different study periods (12 and 36 months before LSM). In the overall study population, no correlation was reported for either HIV-monoinfected patients or subjects with HCV coinfection. Consistently with our results, Merchante et al [15] reported that HIV viremia (binary variable, <200 copies/mL versus >200 copies/mL) at the time of TE is not a factor that is associated with an abnormal LS value (defined as >7.2 kPa in a cross-sectional study including only HIV-monoinfected subjects). Conversely, Mohr et al [22] showed that detectable HIV viremia for a period of more than 2 years was associated with higher LS values in naïve HIV-monoinfected patients; in this study, the subjects were classified as having plasma HIV viremia either greater than 40 copies/mL or undetectable. We were able to evaluate the role of two periods of different length (12 and 36 months) providing the same result. Moreover, the enrolled HIV viremic patients were both naïve and unresponsive to ART; a possible effect of an ART regimen with suboptimal efficacy cannot be excluded. Notably, both in our study and in Mohr’s, there were no correlations found in patients with HIV/HCV coinfection.

The plasma HIV viremia burden may play a role in explaining the differences between these results. Matthews et al [23] showed an association between HIV viremia and LS value in a population of naïve patients who had a median HIV RNA log₁₀ copies of 4.4 (3.9 log₁₀ copies/mL in our study); only 3.9% of the participants had undetectable viremia (the cutoff was set at ≤400 copies/mL, while it was 50 copies/mL in our study). Moreover, higher LS values were correlated with higher log₁₀ HIV RNA values and a threshold effect that was defined as the upper limit of the first two quartiles (19,999 copies/mL) was described; our median values were under this threshold (8979 copies/mL in HIV-monoinfected patients and 962 copies/mL in HIV/HCV-coinfected subjects). A role for the HIV RNA level in determining liver disease was reported by Towner et al [24] in a cohort study involving 20,775 HIV-positive subjects; the authors demonstrated a higher rate of hepatic dysfunction and hepatic dysfunction-related death in the case of plasma HIV RNA>500 copies/mL, while the risk increased with higher HIV RNA levels. A plasma HIV RNA value <500 copies/mL was shown to protect against liver fibrosis progression (determined with FIB-4 and AST-to-platelet ratio index scores) in a cohort of naïve patients who were starting highly active ART [25]; conversely, a median 12% increase in FIB-4 was associated with a 1 log increase in HIV RNA in the study conducted by Forrester et al [26].

The course of liver disease is largely unpredictable on a case-by-case basis, because liver fibrosis is a multifactorial process and the viscoelasticity of the liver may be influenced by inflammatory processes, as demonstrated in post–liver-transplant acute cellular rejection [27]. Our statistical model identified a group of HIV-monoinfected patients with LS values that were discordant with respect to the predicted values; HCV infection had a much stronger hepatic damage capacity than did HIV infection, and this probably justified the absence of coinfected patients among the left and right outliers. The mean BMI was ≥25 in the left outliers (the predicted LS value was higher than or comparable to the observed LS value) and in the right outliers (the observed LS value was higher than the predicted value); increased levels of inflammatory mediators have been described in subjects with HIV who were overweight and this could have perturbed the model [28]. Liver fibrosis is associated with higher BMI values in HIV-monoinfected subjects [29], and we confirmed this correlation using logistic regression in our cohort; however, both left and right outlier subjects were overweight. Almost all right outlier patients (87.5%) had plasma HIV RNA levels that were undetectable in the 12 months before TE, which is in apparent contradiction to the absence of a correlation between the LS value and HIV viremia. Furthermore, HIV RNA is not the only virological parameter that can be used to evaluate HIV infection; cellular HIV DNA constitutes the viral reservoir and is detectable in peripheral mononuclear blood cells in patients with long-term suppressed viremia [30-32]. Recently, HIV DNA was also identified in the hepatic tissue of patients who died while they were on ART and who had undetectable levels of HIV RNA [33].

TE results are currently interpreted as a range in clinical practice, since this provides a more reliable correlation with the degree of fibrosis [34]. We choose to explore the absolute LS value in our HIV-monoinfected patients in order to highlight the differences in a population whose LS values were almost all included in the F0-F1 class of fibrosis (LSM<7.1). No selection bias was evident; our study population had an LSM value comparable to that of previously described cohorts of HIV-positive patients, ranging from 3.9-4.9 kPa [14,15,35]. The main limits of our study are the influence of intra-observer TE reproducibility and the low number of outlier patients. All of the TEs were performed by the same expert operator and only 5 of the 413 patients who were screened (1.2%) were excluded from the study because of an invalid TE, so good reproducibility was expected [36]. One strength of our study was the careful selection of patients; there were no HBsAg-positive subjects included, patients with HCV coinfection were all HCV RNA-positive and almost all of the patients were Caucasian. The independent predictive role of race has scarcely been studied, but Hispanic/Latino ethnicity was independently associated with higher TE scores in HIV patients [23], and HIV coinfection was predictive of early fibrosis in HCV positive African Americans [37].

In conclusion, the present study demonstrated that LS did not correlate with ongoing HIV replication during 12- and 36-month study periods in immunocompetent HIV/HCV-coinfected patients and in HIV-monoinfected subjects.