Occult Hepatitis C Virus Infection in Spanish Patients with Lymphoproliferative Disorders

1Biomedicine Institute of Seville (IBIS), University of Seville, CSIC, University Hospital Virgen del Rocio, Seville, Spain 2Department of Immunology, IBIS, University of Seville, CSIC, University Hospital Virgen del Rocio, Seville, Spain 3Department of Hematology, IBIS, University of Seville, CSIC, University Hospital Virgen del Rocio, Seville, Spain Received date: 03 May 2017; Accepted date: 24 Jul 2017; Published date: 31 Jul 2017.


Introduction
Hepatitis C virus (HCV) infection is a global pandemic.Following primary infection, a minority of individuals clear the infection predominantly via cellular immune mechanisms, whereas the majority become chronically infected [1].
Early after its discovery, it was shown that HCV is also a lymphotropic virus [2] being able to infect and replicate in established B cell lines and primary B lymphocytes [3], as well as in T cell lines and primary naïve T lymphocytes [4].This lymphotropism had various effects, especially on cell development and proliferation [4][5][6].Furthermore, B cells can harbor occult variants that have a poor translational efficiency in hepatocytes, suggesting their extra-hepatic origin [7].Several lympho proliferative disorders (LPD) have been associated with HCV, including B-cell non-Hodgkin lymphoma (B-NHL), between others [8].However, the epidemiological estimates do not take into account the extra hepatic aspects of HCV infection, neither the third outcome: the occult HCV infection (OI) [1,8].
The genomic and antigenomic (replicative) HCV-RNA strands has been found in the liver, serum and peripheral blood mononuclear cells (PBMCs) of patients with antibodies against HCV (anti-HCV), years after spontaneous or treatment-induced resolution of HCV infection [9][10][11][12].These situations are interpreted as OI, which has also been detected in subjects without anti-HCV or HCV-RNA in serum tested by routine methods: patients in whom the etiology of persistently abnormal results of liver-function tests is unknown [13] or population unselected for hepatic disease [14], between others cohorts as heterosexual partners of chronically infected patients or family of patients with OI [15,16].The ultracentrifugation of serum samples, the analysis of immunoglobulin G (IgG) antibody to a dominant HCV core epitope (anti-HCV core), together with another non-invasive assay: test HCV-RNA in PBMCs, have allowed the detection of this form of HCV infection without the need of a liver biopsy [17][18][19][20].However, the possible consequences of this type of infection remain unknown.The purpose of this study was to know the existence of OI in PBMCs and serum from Spanish patients with LPD, without anti-HCV or HCV-RNA in serum tested by routine methods.

Routine detection of HCV, HBV and HIV
HCV-RNA in serum was investigated by Cobas Amplicor HCV test version 5.0, with a lower limit of detection of 15 IU/ml (Roche Diagnostics, Mannheim, Germany).The analysis of anti-HCV in serum was done by the third-generation enzyme immunoassay ADVIA Centaur HCV assay (Siemens Healthcare Diagnostics, Deerfield, USA).HBV surface antigen and HBV core were analyzed by E170 and HIV by Cobas 680 (Roche both).The study was approved by the Ethical Committee of the aforementioned Hospital and conducted according to the Declaration of Helsinki on human experimentation.Informed consent was obtained from all the patients.

HCV RNA detection and quantitation from PBMCs
PBMCs from patients with LPD were isolated from EDTA-treated blood by Ficoll-Hypaque density gradient centrifugation.CD19+ B cells were isolated from PBMCs by positive selection using CD19 Micro Beads and MS Columns (Miltenyi Biotec Inc. Auburn, CA, USA), whereas the others components of the PBMCs, mainly T lymphocytes and monocytes together with NK cells (CD19-fraction), were collected by negative selection in the same sample.Both populations were resuspended in Tripure (Roche) and stored at -80°C.RNA from the CD19+ and CD19-fractions were purified using the Direct-zol™ RNA MiniPrep kit (Zymo Research, Irvine, CA, USA), following manufacturer's instructions.RNA was stored at -80°C after measuring the concentration by spectrophotometry (NanoDrop ND-1000, Wilmington, USA).
Detection of the 5′ non-coding region of the genomic and antigenomic HCV-RNA strands, was performed by a strand-specific real-time RT-PCR, using the Transcriptor First Strand cDNA Synthesis kit (Roche) according to the kit instructions, with 0.5 µg of the total RNA isolated, and 0.5 µmol L-1 of the antisense or sense primers belonging to the HCV untranslated region (UTR) (UTRLC2:5′-CAAGCACCCTATCAGGCAGT-3′;UTRL C1:5′CTTCACGCAGAAAGCGTCTA-3′, respectively).Real-time PCR was done in LightCycler 2.0 (Roche Molecular Biochemicals, Mannheim, Germany), using fluorescence resonance energy transfer (FRET) probes, with the Light Cycler FastStart DNA Master HybProbe Kit (Roche Molecular Biochemicals), as described [18].A standard curve constructed with 10-fold dilutions of synthetic genomic HCV-RNA was used for the quantification of HCV-RNA in PBMCs [21].The sensitivity of this realtime PCR was ten genome copies per reaction.

Enzyme immunoassay to detect anti-HCV core in serum
Analysis of anti-HCV core was done by Kit DIATER ANTI VHC CORE ALTA SENSIBILIDAD (Diater Laboratorio de Diagnóstico y Aplicaciones Terapeúticas (Leganés, Madrid, Spain), following manufacturer's instructions [19] Open Access

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positive and negative controls supplied by the kit) were diluted 1/10 in blocking buffer and pre-incubated for 1 h at 37°C with shaking.After wash twice the 96-well micro titre plate, coated with anti-HCV core, duplicate samples reacted with it for 1h at 37°C.Wells were washed five times with the appropriate buffer and, after removing liquid excess, the conjugated serum was added.After another incubation and wash cycle in the same conditions, the revelation solution was added.Finally, after 30 min incubation at room temperature, dark and shaking, the absorbance values were measured at 405 nm with a reference at 620 nm in the Microplate autoreader EL 309 (Bio-tek instruments, Cultek, Madrid, Spain).The samples were considered non reactives, potentially reactives or reactives following the kit instructions: after validating the assay with absorbance means for blank wells, positive and negative controls, the cut-off point was calculated and finally the absorbance index (AI), dividing the absorbance mean of the sample in duplicate between the cut-off point.Samples with AI ≥ 1.2 are considered reactives, 1.1 ≤ AI ≤ 1.2 are considered potentially reactives and AI ≤ 1.1 non reactives.

Results
HCV-RNA detection and quantitation in the CD19+ cells or CD19fractions from patients with LPD have been summarized in Table 2. Table 3 shows epidemiological and laboratory data of patients with OI.Genomic (positive) HCV-RNA strand was detected in PBMCs of 8 patients (13.33%): one patient diagnosed of HL, 6 B-NHL and another of T-NHL.Six of these 8 patients had sample enough to analyze the HCV-RNA anti genomic (negative) strand too, being detectable it in two of them (2/6=33.33%).Seven of the 60 patients with LPD had HCV transmission risks (familial and professional, Table 2), and 4 of them had OI.With regard to anti-HCV core detection in serum, the HL patient, who had the highest values of HCV viral load of both HCV-RNA strands in the CD19-fraction, was potentially reactive (AI=1.13).In the other patients, an AI lower than 1.1 was obtained, corresponding to non-reactive sample for anti-HCV core detection in serum.

Discussion
The relationship between the classic HCV infection and LPD was documented as consequence of the viral lymphotropism [2,22].However, HCV replicates in the same cells in the classic and occult infection [23,24].As the lymphoproliferation studies have been performed on cohorts of patients chronically infected [8,25,26], the etio pathogenic link between HCV-associated lymphoproliferative disorders may be still undiscovered.In the present work, OI was detected in the 13.33% of patients with LPD, negative for anti-HCV antibodies or HCV-RNA in serum analyzed routinely.
Three general theories have emerged to understand the HCV-induced lymphomagenesis: (a) continuous external stimulation of lymphocyte receptors by viral antigens and consecutive proliferation: (b) HCV replication in B cells with oncogenic effect mediated by intracellular viral proteins and (c) ''hit and run'' theory or permanent B-cell damage caused by a transiently intracellular virus [27].And all these theories could also meet on the OI stage.It has been found a higher expression of low-density lipoprotein receptors involved in PBMCs HCV entry, as well as viral replication, in lymphocytes and monocytes from sustained Open Access
On the other hand detection of high viral loads had been described in B-cells from patients with HCV persistence in PBMCs, which resolved the HCV infection spontaneously or after achieving a sustained virological response [23,24].Furthermore, an enhanced expression of lymphoma genesis-related genes in B-cells from SVR with OI, as recently observed by our group [29].
Assessment of the existence of OI in LPD patients had been documented in Egypt and worldwide in 2012 [30].The authors studied anti-HCV as well as HCV-RNA positive and negative strands from serum and PBMCs in 50 LPD patients, and in 50 apparently healthy volunteers.Anti-HCV and serum HCV-RNA antibodies were detected in 13 out of 50 LPD patients (26%).The HCV-RNA positive strand was detected in the PBMCs from 18 out of 50 LPD patients (36%), among them 10 (20%) patients were negative for serum HCV infection markers, representing the cohort with OI.The HCV-RNA negative strand was undetectable in patients with the positive one.Finally, in two healthy controls (HC), the HCV-RNA positive strand was detected (2/50=4%).So, the occurrence of OI in Egyptian with LPD in comparison with HCV chronically infected patients with LPD and HC were 20, 26 and 4%, respectively; therefore, less incidence than in the HCV chronically infected patients but more that the probability found in the healthy population.
The present work has a limitation due to the lack of internal controls performed in parallel on apparently healthy donors using the same methodology.However, in spite of the lack of studies in Spain, this results are between those found in other European (Italian) cohorts with regard to LPD in patients with chronic HCV infection (19.8%) and the incidence of OI in HC (3.3%) [14,31].In this work a cohort of HC was not included since in previous studies, 72 were analyzed and OI in their PBMCs were not found.Demographic data from these cohorts in comparison with the present one, patients with LPD, are as follows: HC, Gender=39 men and 33 women; age (mean age ± standard deviation)=44.9± 8.56; patients with LPD, Gender=39 men and 21 women; age (mean age ± standard deviation)=47.1 ± 13.85 [15,28,29,32].On the other hand, in 31 sexual partners of chronically HCV infected individuals with HCV transmission risk, (Gender=9 men and 22 women; age (mean age ± standard deviation)=42.74± 9.89), a similar percentage of HCV-RNA presence in PBMCs (13%) than in patients with LPD (13.33%) was observed [15].
In an Iranian cohort, HCV-RNA was found in PBMCs from 2 (1.9%) of the 104 patients with LPD studied, but taking into account the prevalence of HCV infection in general population (<0.5%), the prevalence of OI in LPD patients was approximately 4 times the rate in Iran [33].Kisiel E et al. [34] also analyzed seronegative HCV infection in 77 anti-HCV-negative patients with LPD, studying HCV-RNA by RT-PCR in plasma, PBMCs and bone marrow as well as the presence of viral nonstructural protein 3 (NS3) by immunostaining in PBMCs and bone marrow.HCV-RNA was detected in at least one compartment in 27 (35.1%)patients: in bone marrow in 22 patients (28.6%), in PBMCs in 13 (16.9%)and in plasma in 10 (13%).Viral load in HCV-RNA-positive plasma ranged from 15 to 1.17 x 10 3 IU/mL.NS3 was detected in all but two HCV-RNA-positive bone marrow and one HCV-RNA-positive PBMCs samples [34].So this paper add evidences that HCV can be present among patients with LPD despite the lack of specific antibodies, and highlights the importance to check it in the bone marrow compartment, where the highest HCV-RNA prevalence was found.
Therefore, despite the HCV-RNA negative strand only was studied in the present work together with the Egyptians [30], the discovery of the HCV-RNA positive one is indicative that an OI may exist in patients with LPD.
In the present work, load of genomic and anti genomic HCV-RNA in PBMCs ranged from 2.91 to 986 and 50.4 to 387 copies/µg of total RNA in PBMCs, respectively.Furthermore, the patient with HL (follicular lymphoma), who had the highest values of HCV-RNA positive and negative strands in PBMCs, was potentially reactive for the detection of anti-HCV core in serum by the enzyme immunoassay too.Further studies are required to analyze this phenomenon, to identify these patients as well as to evaluate the possibility that it might be related with the development of lymphoproliferative disorders.

Table 1 :
Epidemiological and laboratory data of the patients.

Table 2 :
OI in patients with LPD.

Table 3 :
Epidemiological and laboratory data of the patients with OI.