Neutrocyte-to-lymphocyte ratio predicts the presence of a replicative hepatitis C virus strand after therapy with direct-acting antivirals

Secondary occult hepatitis C infection (OCI) is defined as the presence of HCV-RNA in liver tissue or peripheral blood mononuclear cells (PBMCs) in anti-HCV-positive patients repeatedly negative for serum HCV-RNA who reached a sustained virological response (SVR) after antiviral therapy [1]. Various types of lymphoid cells were shown to support HCV replication, being an important, long-term reservoir of the virus [1‐3]. Viral infection of PBMCs in chronic hepatitis C (CHC) has a profound effect on immune cell function and carcinogenesis, leading to lymphocyte proliferative disorders, including mixed cryoglobulinemia and B cell non-Hodgkin lymphoma [4, 5]. Also persistence of residual HCV-RNA in PBMCs after therapy in SVR patients is linked with impaired immune responses [6, 7], as well as the risk of progressive liver disease and extrahepatic manifestations of HCV infection [1, 8].

The problem of OCI gained recently much attention with the introduction of interferon-free regiments for the treatment of CHC. Persistence of HCV-RNA in liver tissue after reaching SVR after direct-acting antivirals (DAAs) was shown in patients awaiting liver transplantation and in HCV-infected liver transplant recipients [9, 10]. The presence of HCV-RNA (−) strand, a signature of viral replication, was also documented in the latter group of patients [10].

The general prevalence of OCI after therapy with DAA and the mechanisms leading to viral persistence remain unknown. Viral replication and production of HCV particles occurs in OCI at a very low level, which makes persisting HCV-RNA undetectable with conventional clinical tests of serum samples. IFNL genotype is a known predictor of spontaneous or treatment-induced HCV clearance [11]. We aimed to investigate if basic immunological markers and IFNL genotype can be predictive of viral persistence after therapy with DAAs.

Selected characteristics of individual patients during and after DAA therapy are shown in Table S2. All 42 subjects were HCV-RNA negative in serum 24 weeks and remained negative 12–15 months after EOT. Thirty-one patients (74%) were positive for HCV-RNA in PBMCs, and in 29 (69%) of them we confirmed the presence of HCV-RNA (−) strand. Normalization of ALT at the follow-up was noted in 34 individuals (81%). Neither ALT level nor ALT normalization after therapy correlated with the presence of HCV-RNA (Tables 1, S2; Fig. S2). Treatment with OBV/PTV/r + DSV ± RBV was most effective in eradication of replicative viral RNA strand (Table 1).

In case of 10 patients adverse events occurred during DAA therapy and follow-up (Table S2), including portal vein thrombosis with liver failure, ascites, severe bacterial infections, vasculitis, nephropathy, monoclonal gammopathy, lymphomas and rapidly elevating ALT after treatment (Table S2). In three cases of hepatocellular carcinoma (HCC) diagnosed and treated with radiofrequency ablation before therapy no recurrence was observed during follow-up. Occurrence of adverse events did not correlate with the presence of HCV-RNA in PBMCs (Table 1, Table S3), but it associated with lower lymphocyte counts during and after therapy (Fig. S3).

Polymorphisms rs368234815 ΔG/T and rs12979860 T/C were in a complete linkage disequilibrium (r2 = 1), so further we will refer only to rs12979860. Allele frequencies for rs12979860 (f_C = 0.488; f_T = 0.512) differed significantly from their distribution in European population (f_C = 0.691; f_T = 0.309), with unfavorable, minor T allele being more frequent than in population (Chi² = 16.197; P < 0.0001). There was no correlation between IFNL3 genotype and the presence of HCV-RNA in PBMCs (Table 1; Table S3). Favorable homozygote rs12979860 CC had lower neutrocyte levels at the beginning of therapy (2.2 vs. 3.2 × 10⁹ cells/L; P = 0.024) and after 24 weeks (2.3 vs. 3.3 × 10⁹ cells/L; P = 0.037) (Fig. S4).

Test for matched pairs revealed that for all patients significant fall of lymphocyte number occurred during therapy [1.75 × 10⁹ (0.61–4.5) vs. 1.32 × 10⁹ (0.42–5.36) at EOT; P = 0.009)], and neutrophils remained at the same level (P = 0.090) (Fig. S5A–B). During 12 weeks of therapy NLR, SII and PLR values rose significantly for all individuals (P < 0.0001 for all markers), while MPV decreased (P = 0.001) (Fig. S5C–F). Additionally, SII and PLR remained significantly higher from baseline at the follow-up (P = 0.02 and 0.044, respectively) (Fig. S5D–E).

There were no significant differences in neutrocyte and lymphocyte counts between groups of patients with or without viral RNA in PBMCs (Fig. 1a, b). However, a smaller increase in neutrocyte count between EOT and baseline was observed in patients with HCV-RNA (−) (Table 1). Higher NLR value during and shortly after therapy associated with clearance of replicative HCV-RNA strand (Table 2; Fig. 1c). Among patients without HCV-RNA (−) NLR was significantly higher from baseline at 4, 8, 12 weeks after the start of therapy (P values from sign test 0.027, 0.006 and 0.006, respectively), while in the group with replicative strand NLR was significantly raised from baseline only after 12 weeks (P = 0.001) (Fig. 1c). Also lower SII linked with detected HCV-RNA (−) in PBMC but only at 4 and 24 weeks after the start of therapy (Table S4; Fig. S6A). Association of NLR and SII with the presence of total HCV-RNA in PBMCs was much weaker (Table S2, Table S4). There were no significant differences in PLR or MPV between groups of patients (Table S4; Fig. S6B–C).

This brief report shows high occurrence of occult HCV infection in patients who reached SVR24 after DAAs. In the majority of these subjects we also found a replicative HCV-RNA (−) strand. These results are consistent with observations of Elmasry et al. that while in the liver of CHC patients a 1–3 logs difference in the number of copies is seen in favor of HCV-RNA (+) strand, in OCI the amounts of HCV-RNA (+) and (−) strand are similar [10].

Normalization of ALT was not a predictor of OCI or the presence of HCV-RNA (−). This is in accordance with recent report in which elevated liver enzymes after DAA therapy did not predict persistence of viral RNA [10]. Additionally, OCI has been frequently detected in patients after successful IFN-based therapy having normal levels of liver enzymes [13, 14].

It is now established that genetic variations within IFNL3-IFNL4 gene region impact interferon-stimulated gene expression (ISGs) both in the liver and in PBMCs of CHC patients and are predictors of spontaneous and treatment-induced HCV clearance in both DAA- and IFN-based therapy [15‐17]. However, their impact on development of OCI is still unclear. Elmasry et al. [10] examined 9 liver transplant recipients who did not reach normalization of liver enzymes after DAA therapy. They found OCI in all homozygotes in unfavorable rs12979860 T allele (n = 5), while carriers of C/T and CC alleles did not have HCV-RNA in liver tissue or PBMCs [10]. In another study prevalence of the rs12979860 CC genotype was significantly higher in patients with seronegative OCI (no anti-HCV Abs) than in CHC group. Additionally, among patients with this type of occult infection the presence of rs12979860 CC genotype associated with lower HCV-RNA load in liver tissue [18]. Polymorphisms in the IFNL3-IFNL4 region also link with the presence of HCV-RNA in PBMCs both during chronic HCV infection and in patients who reached SVR after IFN treatment [19, 20]. We found no significant correlation between IFNL genotype and the occurrence of HCV-RNA in PBMCs. This may be due to a small group of patients which is not representative of a whole population, as the unfavorable rs12979860 T allele is significantly more prevalent in the tested group. It was shown that at the beginning of DAA therapy rs12979860 CC carriers exhibit decreased expression of ISGs in peripheral blood and liver [17]. As ISGs represent a diverse group of gene products which can also act as inflammatory cytokines this observation corresponds to lower neutrocyte levels at baseline observed in our study for rs12979860 C homozygotes.

We aimed to evaluate if markers of systemic inflammation, such as NLR, SII, PLR and MPV, which are useful prognostic factors in many chronic diseases, can predict development of OCI after DAAs. It was reported that increased PLR in CHC patients during therapy predicts a good virological response [21]. MPV indicates inflammation and liver damage in CHC patients [22], while SII is a prognostic factor in patients with HCC [23].

Therapy with DAAs is known to evoke dynamic changes in cytokine profile and in the balance between neutrophil and lymphocyte counts, as well as to induce a general inflammatory state [23‐25]. In DAA-treated patients a significantly greater increase in neutrophil counts and decrease in lymphocyte numbers between EOT and baseline associated with later development of HCC after therapy [25]. Viral clearance and SVR induced by DAA therapy is accompanied with downregulation of signaling with type II and type III IFNs, but also with elevation of IFN I cellular pathways, which can promote elimination of the virus [26]. In patients achieving SVR after DAAs ISGs expression in liver and blood at EOT is significantly increased in comparison with baseline [27].

We found that a significant and transient rise in NLR, a marker of systemic inflammation, during and shortly after DAA therapy can predict the clearance of replicating HCV from PBMCs. Additionally, patients without HCV-RNA (−) strand had a significantly greater increase in neutrocyte counts between EOT and baseline. We hypothesize that differences in patient’s immune responsiveness at baseline and in the ability to induce general inflammatory response after viral suppression by DAAs determine subsequent HCV persistence/clearance. Although this systemic inflammation could be beneficial for complete eradication of HCV, it might also favor carcinogenesis [23, 25].

Our study is limited by a small sample size and restricted to patients infected with HCV genotype 1. Therefore, further research on larger cohorts is needed to find if transient rise in NLR could be used as a predictor of viral clearance. Also long-term follow-up studies of patients with residual HCV-RNA should be performed to examine the natural course of secondary occult infection after DAAs and its clinical significance.