Peripheral neuropathy after viral eradication with direct‐acting antivirals in chronic HCV hepatitis: A prospective study

Abstract Background HCV‐related extra‐hepatic complications include peripheral neuropathies, with important prevalence and impact. A recent metanalysis of previous intervention trials concluded for insufficient data to support evidence‐based treatments for this complication. In this longitudinal study, we assessed for the first time prevalence and outcome of neuropathy in a cohort of patients with chronic HCV, before and after direct‐acting antiviral agent (DAA) treatment. Method Ninety‐four patients (mean age 58.5 ± 9.9, infection duration 22.2 ± 6.3 years) without systemic and metabolic diseases, underwent neurological examination and electroneurography studies before (T0) and 10.4 ± 1.7 months after the end of DAA therapy (T1), and cryoglobulins (CG) assessment. Muscle strength was evaluated by Medical Research Council (MRC) score; neuropathic pain, sensory function, disability, quality of life were assessed by validated questionnaires (DN4, NPSI, SSS, INCAT and Euro‐QoL). Results At T0, sensory‐motor neuropathy was detected in 22 patients (23%), reflexes were depressed in 32 (34%) with no association with infection duration, viral load, age, CG. Neuropathic pain (DN4 ≥4) was present in 37 patients (39%). At T1, out of the 22 patients with altered electroneurography, 3 had died or developed HCC, 4 showed normal electroneurography, and nerve amplitude parameters tended to improve in the whole group. Only 11 patients (12%) had depressed reflexes and 10 (11%) DN4 ≥4 (P < .05 compared to T0). Scores for MRC, questionnaires and Euro‐QoL improved significantly (P < .05). Conclusion Our study confirms the high prevalence of clinical and subclinical peripheral sensory‐motor neuropathy in patients with HCV infection and indicates improvement after eradication by DAA. These results support the need for larger intervention studies.


| INTRODUC TI ON
Hepatitis C virus (HCV) infection is a major cause of morbidity and mortality, with a strong socio-economic impact. The WHO estimates that 1.1% of the global population has HCV, with wide geographic distribution and potential underestimation as silent infections progress asymptomatically for years. An estimated 1.4% of the population in the US and an estimated 1.25%-1.75% in Italy has HCV, reaching 20% in people above 70 years of age 1 in some areas. HCV infection is associated with several extrahepatic manifestations that increase morbidity and mortality, decrease quality of life 2,3 and may contraindicate antiviral therapy, especially with interferon α (IFNα).
On the other hand, successful eradication of HCV with IFNα and ribavirin was accompanied by improvement of some of these manifestations, with possible resolution in case of sustained virological response, as in the case of mixed cryoglobulinemia. 4,5 Extrahepatic comorbidities of chronic HCV infection include neurological complications, involving both the central (fatigue, cognitive impairment) and the peripheral nervous system. [6][7][8] Peripheral neuropathies, cryoglobulinemic or non-cryoglobulinemic are the most common neurological complications, with a prevalence of up to 86% of infected patients with, and 43.5% of those without, cryoglobulinemia. Prevalence, however, varies depending on the study population, the definition and method of assessment of neuropathy, including electrophysiological studies and standard questionnaires. 4,8,9 For instance, standard electrophysiological studies detected peripheral neuropathy in 15.3% of patients, subclinical in approximately one-third of them. 9 Prevalence rises to 90% when considering only subjective symptoms, such as paraesthesias, among patient with mixed cryoglobulinemia. 10 There is, at present, insufficient evidence to support treatment of HCV-related neuropathy and therapeutic approaches may differ, depending on the presence of cryoglobulinemia and the different potential pathogenetic mechanisms underlying nerve damage. 11,12 A recent metanalysis of all intervention trials, including treatments with IFNα, ribavirin, corticosteroids, cyclophosphamide, plasma exchange, and rituximab, alone or in combination, failed to show improvement of HCV-associated peripheral neuropathy up to 36 months post-treatment, while demonstrating potential adverse events. 13 Furthermore, there are no reliable studies evaluating treatment of non-cryoglobulinemic neuropathy associated with HCV infection.
A new era started in 2013 with the introduction of direct-acting antiviral agents (DAAs), achieving HCV eradication rates of approximately 95% with reduced side effects compared to previous classical treatments. 14 Recent studies report favourable outcomes for extrahepatic HCV-related complications, 5 including insulin resistance, glycaemic control 15 or endothelial function and cardiovascular morbidity. 16,17 However, the impact of HCV eradication achieved by DAAs on HCV-related neuropathies has not been explored systematically, and some data are available only in the context of cryoglobulinemic vasculitis. 5 Two case reports of greatly improved neuropathy after diagnosis and eradication of hitherto ignored HCV infection are suggestive of favourable outcomes of DAA therapy on this complication. 18,19 In the present study, we aimed to assess the prevalence of peripheral neuropathy associated with HCV-infection with and without cryoglobulinemia, and to evaluate prospectively the effects of HCV eradication by DAAs, using a global assessment by standard nerve conduction studies, neurological examination, together with validated questionnaires exploring neurological motor and sensory symptoms, disability and quality of life.   The Douleur Neuropathique 4 (DN4) 26 tool evaluates neuropathic pain through 10 items: 7 evaluating pain quality as defined by the patient and 3 based upon clinical assessment of hypoaesthesia to touch and pinprick and allodynia. Total score derives from the sum of all 10 items and the cut-off value for neuropathic pain diagnosis is 4/10. 26 DN4 has 82%-83% sensitivity, and 81%-90% specificity. 26,27 The Neuropathic Pain Symptom Inventory (NPSI) 28 is a selfadministered questionnaire assessing spontaneous ongoing or paroxysmal pain, evoked pain and dysesthesia/paraesthesia. It is structured in 12 items, 10 on different symptoms, and 2 on duration, each quantified on a numerical scale ranging 0-10; total score was recorded.

| Assessment of neuropathy and quality of life
The Sensory Sum Score (SSS) 29 comprises pin prick and vibration sense plus a two point discrimination value in the arms and legs, and ranges from 0 ("normal sensation") to 20 ("most severe sensory deficit").

The Inflammatory Neuropathy Cause and Treatment (INCAT) 30
tool measures disability of upper and lower limbs, testing the ability to use either arm for purposeful movements and to walk without support or wheelchair, respectively. Scores ranged from 0 (no disability) to 12 (severe disability).
Quality of life was evaluated using the Euro-QoL, 31 based upon self-evaluation of health-related quality of life assessing mobility, self-care, usual activities, pain/discomfort, and anxiety/depression, each with 3 levels of increasing severity. Index is then calculated subtracting from 1, the best health state, standard coefficients that increase with gravity.
Investigators studying clinical features were unaware of electrophysiology and lab testing results, as were the other investigators of the clinical data.
At T1, all patients were re-examined and questionnaires administered, and only those with abnormal electroneurography at baseline underwent a new electrophysiological study.

| Statistical analysis
Data are shown as absolute and relative (%) frequencies for categorical data and mean ± SD for continuous variables.

Mc Nemar test for categorical variables and paired t test or
Wilcoxon signed-rank test, as appropriate, were carried out to detect significant differences between baseline and follow-up data for neurological characteristics of all patients and for electrophysiological data for patients who showed abnormal results at baseline.
Chi-square test for categorical variables and t test for continuous variables, or Wilcoxon rank-sum test in case of nonparametric distribution, were performed to assess whether significant differences for demographic and clinical data could be found between patients with and without abnormal electrophysiological results at baseline. For all tests, a P-value of less than 5% was considered significant.
All analyses were performed with Stata 14.

| HCV-related neuropathy
Ninety-four patients were recruited at baseline (T0) and their clinical characteristics are summarised in Table 1  There was a trend towards higher prevalence of altered nerve conduction studies, worse amplitude parameters (significantly different for median nerve CMAP amplitude, 6.7 ± 2.4 vs 8.4 ± 1.9 mV, P = .001, and peroneal nerve CMAP amplitude, 4.8 ± 2.2 vs 6.1 ± 2.1 mV, P = .012) and worse neurological symptoms in CG+compared to CG-patients, with only SSS reaching a statistically significant difference (P = .031) ( Table 3).   When considering the presence of cryoglobulinemia, a significant improvement was detected in both groups (CG+and CG-) from T0 to T1 for all neurological scores. The improvement in the CG+patient subgroup was also significantly higher than the one in the CG-subgroup for neuropathic pain at DN4 (Table 5).

| D ISCUSS I ON
Extrahepatic manifestations are responsible for a portion of the morbidity and mortality related to HCV infections, and have even been regarded as contraindications to treatment in the era of IFNbased therapies, or a matter of concern for drug-drug interactions.
Moreover, studies exploring the effects of successful antiviral thera-  .792 Note: Data are expressed as n (%), or mean ± SD. nerve conduction velocity was detected, possibly reflecting axonal re-growth preceding myelination. In fact, it is established that regenerated, still non myelinated fibres, have got a low conduction velocity undetectable by standard nerve conduction studies. 39,40 Notably, significant neurological improvement was detected also in GC+patients, who presented with worse sensory impairment, despite the fact that cryoglobulins are a negative predictive factor for neuropathy, associated with more severe involvement at histometrical analysis, 8 reflecting additional pathogenetic mechanisms and, possibly, inducing permanent nerve damage. 8,41 There is a growing body of literature on the association between HCV infection and reduced quality of life, impacting all aspects of function. 3     brain microvascular endothelial cells, with release of infectious virus, conformational changes of endothelium, viral and cytokine passage across the blood-brain barrier, together with microglial activation inducing a state of neuroinflammation. 47,48 Furthermore, recent studies indicate that viral eradication with DAAs is associated with a significant improvement in endothelial function, vascular distensibility, reduction in insulin resistance and improved glycaemic control. [15][16][17]48,49 Improved metabolic and vascular functions could thus involve nerve microvasculature, preventing ischaemic damage, curbing endothelial activation and the local inflammatory response. 43 According to international guidelines, from June 2018 all patients with HCV infection should be considered for treatment with DAAs, prioritizing those with symptomatic cryoglobulinemic vasculitis, extensive liver fibrosis and stage 4-5 CKD. 36,50 In line with this recommendation, our report strengthens the indications for early antiviral therapy, independently of the severity of liver disease, on the basis of the risk of developing serious extrahepatic complications, including neuropathy, with a potentially rapidly progressive course. 18,19 The present study has strengths and limitations. The number of subjects examined prospectively is the largest assessed so far for neurological implications of HCV eradication, in a single centre and regardless of cryoglobulinemia. Limitations include lack of assessment of small sensory fibres, which requires more sophisticated tests as pain related-evoked potential and skin biopsy, and the lack of a control group, ethically unconceivable.
This work adds to recent reports of significant benefits after successful eradication of HCV by well tolerated DAA regimens, broadening the spectrum of patients eligible for therapy; HCV-related neuropathy should be considered a major indication for treatment even in the absence of liver disease. Long term follow-up studies exploring whether sustained virological response is associated with wider, further neurological improvement are welcome.

ACK N OWLED G EM ENTS
The authors thank Erdita Peci, BS for support in electrophysiological studies.

CO N FLI C T S O F I NTE R E S T
None of the authors has any conflict of interest to disclose.

AUTH O R CO NTR I B UTI O N S
MMZ was responsible for conception and design of the study, clinical examination, data analysis and wrote the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.