hepatitis c virus and human t cell lymphotropic virus type 1 co infection impact on liver disease virological markers and neurological outcomes

Accepted Manuscript Title: Hepatitis C virus (HCV) and human T-cell lymphotropic virus type (HTLV-1) coinfection: Impact on liver disease, virological markers and neurological outcome Authors: Ot´avio M Esp´ındola, Alexandre G Vizzoni, Elisabeth Lampe, Maria Jos´e Andrada-Serpa, Abelardo Q.C Ara´ujo, Ana Claudia C Leite PII: DOI: Reference: S1201-9712(17)30040-1 http://dx.doi.org/doi:10.1016/j.ijid.2017.01.037 IJID 2862 To appear in: International Journal of Infectious Diseases Received date: Revised date: Accepted date: 14-9-2016 26-1-2017 27-1-2017 Please cite this article as: Esp´ındola Ot´avio M, Vizzoni Alexandre G, Lampe Elisabeth, Andrada-Serpa Maria Jos´e, Ara´ujo Abelardo QC, Leite Ana Claudia C.Hepatitis C virus (HCV) and human T-cell lymphotropic virus type (HTLV-1) coinfection: Impact on liver disease, virological markers and neurological outcome.International Journal of Infectious Diseases http://dx.doi.org/10.1016/j.ijid.2017.01.037 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Hepatitis C virus (HCV) and human T-cell lymphotropic virus type (HTLV-1) coinfection: Impact on liver disease, virological markers and neurological outcome Otávio M Espíndolaa,1, Alexandre G Vizzonia,1, Elisabeth Lampeb, Maria José Andrada-Serpaa, Abelardo Q C Araújoa, Ana Claudia C Leitea a- Laboratory for Clinical Research in Neuroinfections, Evandro Chagas National Institute for Infectious Diseases (INI) – Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro, Brazil; b- Laboratory of Viral Hepatitis, Oswaldo Cruz Institute (IOC) – FIOCRUZ, Rio de Janeiro, Brazil 1- Otávio M Espíndola and Alexandre G Vizzoni contributed equally Corresponding author: A.C.C Leite LAPCLIN-Neuro – INI/FIOCRUZ, Avenida Brasil, 4365 – Manguinhos – Rio de Janeiro/RJ – Brazil ZIP code: 21040-900 Phone: +55(21)3865-9543 E-mail: ana.leite@ini.fiocruz.br Highlights  Frequency of neurological abnormalities is unaltered during HCV/HTLV-1 coinfection  Hepatic damage is reduced during HCV/HTLV-1 infection, even without HCV clearance  Coinfection with HCV does not change HTLV-1-directed CD4+ T-cell proliferation  HCV-related thrombocytopenia is reversed during coinfection with HTLV-1 ABSTRACT Objectives: HTLV-1 infection is associated with neurological abnormalities, such as HTLV1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and peripheral neuropathy (PN) HCV infection is the leading cause of chronic liver disease worldwide, and causes PN in approximately 9% of patients Because the interplay between these potentially neuropathogenic viruses in a same individual is still poorly understood we evaluated clinical and laboratory outcomes in coinfected patients and compared them with controls Methods: The prevalence of neurological and laboratorial abnormalities was evaluated in HCV/HTLV1-coinfected patients (n=50), and in subjects with single HCV (n=46) or HTLV-1 (n=150) infection Results: A higher frequency of isolated PN was present in HCV-infected patients and this was not associated with cryoglobulinemia No difference was found in the frequency of PN or HAM/TSP when compared coinfected with singly infected individuals Hepatic involvement was present in HCV-infected subjects, as shown by increased levels of serum ALT, AST, GGT and bilirubin, in addition to thrombocytopenia On the other hand, HCV/HTLV-1-coinfected individuals presented with a better prognosis for hepatic involvement when compared with singly HCV-infected subjects Conclusions: Our data suggest that HCV/HTLV-1 coinfection does not mutualistically alter the outcome of neurological manifestations Nonetheless, changes in the immunological environment induced by HTLV-1 infection could lead to a reduction in hepatic damage, even without significant HCV clearance Keywords: HCV; HTLV-1; Coinfection; Hepatic involvement; Peripheral neuropathy INTRODUCTION HTLV-1 infects 5−10 million people worldwide, being endemic in Japan, the Caribbean, Sub-Saharan Africa, the Middle East and South America, particularly in Brazil.1 HTLV-1 causes a persistent infection of CD4+ and CD8+ T-cells that leads to diverse changes in the immune response,2 and can be associated with the development of adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/Tropical spastic paraparesis (HAM/TSP).3 In addition to its pathogenic role in these diseases, HTLV-1 has been also associated with other immune-mediated conditions, such as uveitis, polymyositis, chronic inflammatory arthropathy and increased susceptibility to some opportunistic infections.4 Most of HTLV-1-infected individuals remain asymptomatic carriers (ACs) and only 0.5−5% of them develop disease.5 Several studies have suggested that HTLV-1 is associated with a wider spectrum of neurological manifestations that not fulfill the diagnostic criteria for HAM/TSP.3 These symptoms and conditions may later progress to classical HAM/TSP or remain as isolated neurological syndromes, such as sensory neuropathy,6 and bladder dysfunction,8 erectile dysfunction,9 amyotrophic lateral sclerosis-like syndrome,10 mild cognitive deficits11 and more rarely, motor neuropathies.12 Events or mechanisms that trigger the neurological involvement in HTLV-1 are still unknown However, a high HTLV-1 proviral load (PVL) in peripheral blood has been associated to the development of neurological abnormalities.13 HCV infection is the leading cause of chronic liver disease worldwide, and it is estimated that about 185 million individuals are or have been infected throughout the world.14 The average prevalence of HCV seropositivity in Brazil is around 1.23% among blood donors, and in Rio de Janeiro the estimated prevalence is 2.6%.15 The majority of HCVinfected individuals establish a chronic infection, and 74−86% present detectable viremia in peripheral blood In 25−30% of cases chronic infection can progress to severe complications, such as chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC).16 It is also estimated that 40−74% of HCV-infected individuals can develop at least one extrahepatic manifestation, such as mixed cryoglobulinemia, membranoproliferative glomerulonephritis, sicca syndrome, and porphyria cutanea tarda, among others.17 Peripheral neuropathy (PN) can be found in 9% of patients with chronic HCV infection, regardless the presence of cryoglobulinemia.18 Involvement of the central nervous system (CNS),19 and 20 the peripheral nervous system (PNS)21 and, less frequently, the autonomic nervous system, have also been documented, either as a direct manifestation of the infection or as a paraneoplastic presentation in cases of HCC.22 The HCV/HTLV-1 coinfection may be common, particularly in countries where these viruses are endemic, since they share similar modes of transmission This is especially true in certain population groups, such as in persons who have unprotected sex habits, intravenous drug users and individuals with a history of blood transfusion.23 and 24 The cellular immune response apparently play an important role not only in the viral clearance in both infections25 and 26, but also in the immunopathogenesis of the hepatic lesion in HCV infection16 and in the neurological involvement in HTLV-1 infection.3 The interplay between those two neuropathogenic viruses in a same individual is still poorly understood It has been shown by some that, when compared to HCV-monoinfected patients, the HCV/HTLV-1 coinfection has a synergistic effect on incident liver disease, as well as on deaths from HCC.23 However, other groups have postulated that HTLV-1 infection can attenuate hepatic damage, as shown by lower serum levels of aminotransferases and lower degree of liver fibrosis 27 and 28 Similar results were observed in a study with triple HIV, HCV and HTLV-1 coinfection, in which HTLV-1-infected patients presented better outcomes for HCV-associated liver fibrosis.26 Therefore, in order to give new insights about the interactions between HCV and HTLV-1 infections, we evaluated abnormalities on hepatic parameters and the outcome of neurological abnormalities during HCV/HTLV-1 coinfection MATERIAL AND METHODS 2.1 Patients and data collection This cross-sectional study was conducted from 2008 to 2010 at the Evandro Chagas National Institute for Infectious Diseases (INI/FIOCRUZ), Rio de Janeiro, Brazil The study was approved by the local ethics committee (INI 2009/0028.0.009.000.08), and informed written consent was taken from all patients submitted to the research protocol HTLV-1infected individuals (n=150) were randomly selected from a cohort of 650 subjects regularly attending the outpatient clinic at INI/FIOCRUZ HCV/HTLV-1-coinfected patients (n=50) from this same cohort were enrolled in the study when inclusion criteria were fulfilled HCVinfected individuals (n=46) were randomly selected from outpatient cohorts followed at the Gaffrée & Guinle University Hospital (HUGG-UNIRIO), Rio de Janeiro, and at the Oswaldo Cruz Institute (IOC/FIOCRUZ) Individuals with concurrent infection with HIV, HTLV-2, syphilis, and HBV, or presenting conditions that could by themselves cause PN (such as vitamin B12 deficiency, chronic use of potentially neurotoxic drugs, diabetes, leprosy, and renal or genetic diseases) were excluded from this study 2.2 Neurological evaluation Patients were classified as neurological asymptomatic or as HAM/TSP according to the modified World Health Organization’s criteria, as previously described.29 HTLV-1infected patients with urinary urgency/incontinency/retention, and with an abnormal urodynamic evaluation, but without PNS or CNS involvement, were classified as presenting an isolated neurogenic bladder.8 Due to the close relationship between HCV infection and PN in literature,21 a full evaluation of the PNS was prioritized The diagnosis of PN was done when one or more of the following clinical criteria were fulfilled: numbness, burning sensations, pain in a stocking and glove distribution, distal weakness, and diminished or absent deep reflexes with or without distal muscular atrophy In the electromyography (EMG) and nerve conduction studies (NCS), the following diagnoses were considered according to classical criteria:30 polyneuropathy or multiple mononeuropathy Presence of specific histological abnormalities in sural nerve biopsy was also considered as a hallmark of pathological involvement of the peripheral nerve Isolated PN was identified in patients presenting signs and/or symptoms of PN and an abnormal NCS/EMG and/or abnormalities in the sural nerve biopsy; or when clinical criteria of PN were present without confirmation by NCS/EMG and/or histological evaluation of the peripheral nerve.12 2.3 Clinical and laboratory data Sociodemographic data such as gender, age, intravenous drug use (IDU), alcohol consumption, self-reported history of blood transfusion, number of sexual partners and practice of unprotected sex were assessed Peripheral blood was collected in EDTA and used on the same day for DNA extraction, for subsequent HTLV-1 proviral load quantification, and in determining complete blood counts, and CD4+ and CD8+ T-cells counts by flow cytometry (FACSCalibur, Becton Dickinson) Plasma was obtained from peripheral blood in EDTA by centrifugation at 1,500  g for 10 minutes and used for assessment of cryoglobulins as previously described.31 Serum samples were used within four hours for quantification of aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), and total bilirubin levels, or aliquoted and stored at -80C for further analysis Liver fibrosis and cirrhosis were predicted using APRI (AST to platelet count ratio index).32 HTLV-1 diagnosis was based on the detection of specific anti-HTLV-1 antibodies in serum by enzyme immunoassay (EIA) (Anti-HTLV-I ∕ II Sym Solution, Symbiosys, São Paulo, Brazil) and confirmed by Western blot (HTLV Blot 2.4, Genelabs Technologies, Singapore) The diagnosis of HCV infection was performed by a third-generation EIA (ETIAB-HCVK-4, Diasorin, Vercelli, Italy), according to the manufacturer’s recommendations, and confirmed by HCV RNA genomic amplification (Amplicor ® HCV Test, v2.0, Roche Diagnostics, France) HCV genotyping was performed using the Versant™ HCV Genotype Assay - LiPA (Bayer Corporation, Tarrytown, NY, USA), which can discriminate HCV genotypes to The quantification of HCV viral load was performed using the Cobas™ Amplicor HCV Monitor Test, v2.0 system (Roche Diagnostics, France), according to manufacturer’s instructions HTLV-1 PVL was determined by quantitative PCR, as previously described,13 and shown as frequency of HTLV-1-infected cells in peripheral blood leukocytes (PBL) 2.4 Statistical Analysis Statistical analysis was performed using SPSS software v.20 Comparison of categorical variables among groups was performed by Pearson chi-square (2) test, or Fisher’s exact test when cell size was fewer than five Normal distribution of continuous variables was evaluated by Kolmogorov-Smirnov test Comparison of continuous variables was performed by Student’s t test or Mann-Whitney test Multiple comparisons were carried out by One-way ANOVA (parametric) or Kruskal-Wallis test (non-parametric) Pairwise comparisons were run in R software v.3.2.5 using Fisher’s exact test or Mann-Whitney test with Bonferroni correction Results with p