Occult hepatitis C infection identified in injection drug users with direct antiviral agents therapy and spontaneous resolution of hepatitis C virus infection

Highlights • Occult hepatitis C infection (OCI) identified in injection drug users (IDUs) with direct-acting antivirals (DAAs) therapy and HCV spontaneous resolution) by droplet digital PCR (ddPCR).• OCI IDUs with DAAs therapy might not be entirely cured.• OCI IDUs without DAAs therapy identified due to tested PBMCs samples.• Hight percentage of HCV-RNA detected in red blood cells (RBCs).• HCV/OCI identification with PBMCs and RBCs samples as a predictor.


Introduction
Occult hepatitis C infection (OCI) is characterized by the detection of hepatitis C virus (HCV) ribonucleic acid (RNA) in hepatocytes and peripheral blood mononuclear cells (PBMCs) without detection in serum by conventional PCR assays (Castillo et al., 2004).
The current understanding of OCI and its clinical implications remain unclear, although, this infection has been described in seropositive and/or seronegative patients with chronic liver disease, coinfections or comorbidities (Austria and Wu, 2018;Hedayati-Moghaddam et al., 2021;Naghdi et al., 2017;Frias et al., 2019;Wroblewska et al., 2021). Data describing that OCI could be present in many individuals who have "cleared" the virus spontaneously from their serum was also previously described (Yousif et al., 2018;Wang et al., 2019). Moreover, OCI was described in population with risk factors, such as in subjects with tattoos, acupuncture and apparently in healthy population (Martinez-Rodriguez et al., 2018;Helaly et al., 2017). Furthermore, it was also previously described in injection drug users (IDUs), and being this population a high-risk group for blood-borne infectious diseases transmission, OCI positive individuals could lead to HCV transmission Donyavi et al., 2019;Sugden et al., 2013;Enkelmann et al., 2020). The role of HCV positive in no injection drug users (NIDUs), such as individuals that inhales crack cocaine, powder cocaine, consumes methamphetamines or heroin, could lead to HCV transmission if the sharing of their used pipes, straws or tubing were blood-contaminated, and OCI was also previously described in this population (Scheinmann et al., 2007;Van den Berg et al., 2009;Schuch-Goi et al., 2017). The therapy with direct-acting antivirals (DAAs) of chronic hepatitis C markedly improved sustained virological response (SVR) rates, although OCI has been described in several conditions and after DAAs therapy (Yousif et al., 2018;Wang et al., 2019;Elmasry et al., 2017;Mohamed et al., 2019;Mekky et al., 2019). Likewise, HCV-cure in subjects with HCV or OCI infection that presented SVR after DAAs therapy may not be entirely valid (Attar and Van Thiel, 2015;Lybeck et al., 2019). Studies reporting patients' treatment failure after DAAs therapy that used e.g. glecaprevir/pibrentasvir (GP), ledipasvir/sofosbuvir (LED/SOF), sofosbuvir/velpatasvir (SOF/VEL) and elbasvir/grazoprevir (ELB/GRZ) antivirals were previously described (Attar and Van Thiel, 2015;Lybeck et al., 2019;Ghany et al., 2020;Zahid et al., 2022).
Recently we have presented preliminary data regarding OCI patients treated or not-treated with DAAs (Silva et al., 2022) and also their possibility of HCV/OCI transmission (Silva et al., 2023), and here we aimed to evaluate OCI in drug and non-drug users (NDUs), including patients who achieved SVR after DAAs therapy (AVP group) and with spontaneous resolution of HCV infection (NAVP group). Serum and PBMCs samples were screened for HCV/OCI-RNA detection by droplet digital PCR (ddPCR), as this is a ultra-sensitive technology for low RNA viral load detection (Frias et al., 2019;Silva et al., 2023). Additionally, plasma and red blood cells (RBCs) patients' samples were also screened for HCV/OCI-RNA detection by ddPCR.

Study groups
Patients with HCV infection from the Coimbra Hospital and University Center (CHUC), between 2019 and 2021, were included in this study. All patients were anti-HCV positive and tested negative for hepatitis B virus and human immunodeficiency virus (HIV). A total of 44 patients (IDUs, NIDUs and NDUs) were included and divided in the following study groups: AVP -24 patients (22 IDUs and 2 NDUs) with 8 or 12 weeks DAAs therapy who achieved an SVR-12; NAVP -13 patients (4 IDUs, 6 NIDUs and 3 NDUs) with HCV spontaneous resolution. An HCV positive control group (CPP) (HCV RNA detected by real time PCR (RT-PCR)), including 7 NIDUs patients was also considered. Key clinicopathological data of these patients were collected and summed up in Table 1. Patients with confirmed HCV infection were treated with GP, LED/SOF, SOF/VEL or ELB/GRZ, according to the EASL recommendations, and all of them achieved an SVR (12 weeks) determined by HCV negative results by RT-PCR. The CHUC Ethics Committee approved the study (registration number CHUC-122-18). Written informed consent was provided by all the patients.

Samples collection
Blood samples of all patients/groups were collected in dry and in lithium heparin tubes. Serum was recovered from the dry tubes as previously described (Costa-Matos et al., 2013). PBMCs were isolated from the lithium heparin tubes using lymphoprep™ (Alere Technologies AS, Norway) following the manufactures instructions, and recovered PBMCs were resuspended in 200 µL of water molecular grade (G-Biosciences, USA) and also in 200 µL of roswell park memorial institute (RPMI) 1640 culture medium (Thermo Fisher Scientific, USA) supplemented with 50% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA) and 20% dimethyl sulfoxide (Thermo Fisher Scientific, USA) as previously described (Silva et al., 2023). Plasma and RBCs were also collected as previously described (Silva et al., 2023). All samples were stored at − 80 • C. After, serum, plasma, PBMCs resuspended in water molecular grade and RBCs resuspended in water molecular grade patients/groups samples were evaluated for the presence of HCV/OCI-RNA by ddPCR. OCI patients identification was done considering ddPCR serum negative and PBMCs positive results as previously described (Castillo et al., 2004;Silva et al., 2022;Silva et al., 2023).

RNA extraction, cDNA synthesis and ddPCR
Total RNA was extracted from 250 µL of serum, plasma, PBMCs resuspended in water molecular grade and RBCs resuspended in water molecular grade samples of all patients /groups using TRI Reagent LS (Sigma-Aldrich, Germany) as previously described (Silva et al., 2012;Silva et al., 2015) and using the RNeasy Mini Kit (Qiagen, Germany)

OCI
Occult Male 47 IDUs -- Male 41 IDUs -- Silva et al. Virus Research 329 (2023) 199104 following the manufactures instructions and as previously described (Silva et al., 2023). The cDNA synthesis was performed using random hexamers included in the Xpert cDNA synthesis kit (GRISP, Portugal) following the manufactures instructions as previously described (Silva et al., 2023). DdPCR was performed as previously described (Silva et al., 2023). Briefly, HCV/OCI-RNA was detected for the HCV core region using sense 5 ′ -GCGTTAGTAYGAGTGTYG and antisense 5 ′ -CRATTCCGGTGTACTCAC primers, and FAM-labeled HCV probe (5 ′ -FAM-CCGCAGACCACTATGGCTC-BHQ1-3 ′ ) (Frias et al., 2019;Silva et al., 2023). The reaction mix contained 10 μL of ddPCR™ Supermix for Probes (Bio-Rad, USA), 900 nM of each primer, 250 nM of FAM-labeled HCV probe and 2 μL of template in a final volume of 22 μL. The reactions mix were placed on a Bio-Rad QX200 Droplet Digital PCR System for droplets generation following the manufactures instructions, and then HCV/OCI-RNA amplification was performed on a Bio-Rad Thermal Cycler C1000 (Bio-Rad, USA) under the cycling conditions of 95 • C for 10 min, 45 cycles at 94 • C for 30 s and 55 • C for 1 min and 98 • C for 10 min. After, the plate was read on a Bio-Rad QX200™ Droplet Reader for droplets analysis, and data was analysed using the Bio-Rad QuantaSoft™ Analysis Pro-Software v. 1.0.596 following manufactures instructions. The fluorescence amplitude threshold was automatically adjusted in individual wells. At end-point reactions the droplets are scored as positive or negative attending to the number of observed accepted droplets (10.000 or greater) and these values are used to calculate the HCV/OCI-RNA concentration using binomial Poisson statistics. Attending that ddPCR permits the absolute count of genome copies in individual wells without the requirement of an experimental or established standard curve, here, we considered the lower and the higher limits for HCV/OCI-RNA detection of 0.22 (PBMCs) and 127.34 (serum) copies/µL, respectively, as these were the lower and the higher values achieved in the analyzed blood samples.

Statistical analysis
Frequencies, percentages, and means were used for descriptive analysis in study group samples in Microsoft® Excel® 2016 MSO. Normal distribution was evaluated with Kolmogorov-Smirnov test. Differences in values were evaluated using Mann-Whitney test. For multiple comparisons, one-way ANOVA with Dunnett's multiple comparison test was used. Statistical analysis was performed with the SPSS (Statistics Package for Social Sciences) software version 28.0. Graphs were developed with GraphPad Prism 7.01. Significant levels were set at p<0.05 for all statistical analysis.
In total analysed blood samples, HCV/OCI-RNA was detected in 65.9% of the PBMCs, 63.6% of the serum, 61.4% of the RBCs and 38.6% of the plasma samples within the lower and the higher limits of HCV/ OCI-RNA detection considered for ddPCR assay in this study, that was 0.22 (PBMCs) to 127.34 (serum) copies/µL ( Fig. 1 and Table 2). These values were validated by the achieved number of positive and negative droplets relatively to the obtained number of accepted droplets, as well as, by the obtained values for the binomial Poisson statistics achieved in each run ( Fig. 1 and Table 2). The concentration of HCV/OCI-RNA detected in analysed blood samples and OCI patients' identification by ddPCR are shown in Table 3. Blood samples fluorescence 1D amplitude plots are shown in Fig. S1.  Within each group, 83.3% of HCV/OCI-RNA was detected in RBCs, 79.2% in PBMCs, 70.8% in serum and 12.5% in plasma samples of the patients in the AVP group. In patients of the NAVP group 53.9% was detected in serum, plasma and PBMCs and 38.5% in RBCs samples. In patients of the CPP group, 100% was detected in plasma, 57.1% in serum, 42.9% in PBMCs and 28.6% in RBCs samples. In total, higher values statistically significant of HCV/OCI-RNA were detected in RBCs samples of the patients in the AVP group comparatively to the NAVP (p<0.01) and CPP (p < 0.05) patients' groups by ddPCR, although for the other blood samples no statistically significant differences were shown (Fig. 2).

Discussion
OCI is characterized by the detection of HCV/OCI in PBMCs samples of the patients without detection in serum samples by conventional PCR assays (Castillo et al., 2004;Austria and Wu, 2018;Wroblewska et al., 2021). We have recently performed two studies in OCI where we have characterized OCI patients, considering serum negative and PBMCs positive results by ddPCR, in different study groups and also their possibility of HCV/OCI transmission (Silva et al., 2022;Silva et al., 2023). The OCI was identified in HCV negative patients (CNP group), patients who spontaneously eliminate HCV (SEP group) and in patients who achieved a SVR after DAAs therapy (AVP group) (Silva et al., 2022). Here, we evaluate the presence of OCI in IDUs, NIDUs and NDUs patients in the AVP and NAVP groups by ddPCR, considering also serum negative and PBMCs positive results. Additionally, plasma and RBCs samples of the patients in both groups were also screened for HCV/OCI-RNA detection by ddPCR.
Taking in consideration our results we suggest that the patients in the AVP group might not be entirely cured, and studies reporting treatment failures after GP, LED/SOF, SOF/VEL and ELB/GRZ were previously described (Attar and Van Thiel, 2015;Lybeck et al., 2019;Ghany et al., 2020;Zahid et al., 2022). Our findings questioning the higher DAAs effectiveness described in the literature, that carefully deserves investigation about the meaning of this discrepancy and related clinical significance.
Considering the patients in the NAVP group, obtained results suggest that tested patients at clinical evaluation time were not identified as HCV positive by RT-PCR, probably as just serum samples were screened at that time. These results efforts attention in the currently serum samples screening for HCV diagnose, and also efforts the OCI definition when the screening of PBMCs samples for HCV/OCI-RNA detection was considered.
Furthermore, in total HCV/OCI-RNA was detected in 70.8%, 12.5%, 79.2% and 83.3% of the serum, plasma, PBMCs and RBCs samples, respectively. Higher values, statistically significant, of HCV/OCI-RNA were achieved in RBCs samples of the patients in the AVP, comparatively to patients in the NAVP (p<0.01) and CPP (p < 0.05) groups, by ddPCR, and the detection of HCV in RBCs was previously described (Schmidt et al., 1997;Lotz et al., 2002;Simon et al., 2003). These results Table 2 HCV/OCI-RNA detected in serum of patient 8 and in PBMCs of patient 10 in the AVP group by ddPCR. High (127.34 copies//µL, serum) and low (0.22, PBMCs) limit values of HCV/OCI-RNA detection were established for the ddPCR assay. suggest that RBCs, as PBMCs (Hanno et al., 2014), could be a predictor for HCV/OCI identification in future. Moreover, overall results shown also that ddPCR could be a more sensitive technology for HCV/OCI-RNA detection, as a range of 0.22 to 127.34 copies/µL were able to be detected.
In conclusion, OCI was identified in IDUs patients in the AVP and NAVP groups by ddPCR, suggesting that OCI patients in the AVP group might not have a total viral eradication, and that OCI patients in the NAVP group were not identified at clinical evaluation time due probably as just serum samples were tested at that time. HCV-RNA was also detected in no OCI patients in the AVP and NAVP groups. Overall results suggest that the HCV/OCI identification in patients with sustained viral response after DAAs therapy and those who spontaneously cleared the virus should be performed more accurately in future, as well as, in the diagnose of patients suspected of being infected with the virus. Additionally, PBMCs and RBCs samples are suggested as predictors for HCV/ OCI diagnosis and management in future preventing HCV/OCI transmission. The epidemiological and clinical meaning of this findings deserves further investigation. HCV, hepatitis C virus; OCI, occult hepatitis C infection; AVP, patients who achieved a SVR after DAAs treatment, 8-12 weeks; NAVP, patients without DAAs therapy; CPP, patients HCV positive; PBMCs, peripheral blood mononuclear cells; RBCs, red blood cells; OCI, occult hepatitis C infection; N/D, not determined. *(+) -OCI patient.

Funding
This work was supported by the Foundation for Science and Technology (FCT) under the grant number PTDC/SAU-SER/30,788/2017, FEDER.

Author contributions
Eliane Silva and Armando Carvalho designed the study. Armando Carvalho, Adélia Simão, João Madaleno and Bernardo Canhão participated in the patient's recruitment, data collection, and data interpretation and all together with Eliane Silva participated in the sample's collection. Eliane Silva performed the ddPCR at the Cancer Biology & Epigenetics Group, Research Center of IPO Porto. Sara Marques and Bárbara Leal performed the statistical analysis. Eliane Silva wrote the paper with Sara Marques support. All authors critically reviewed the manuscript and approved the final version of the manuscript for publication.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
Data will be made available on request.

Supplementary materials
Supplementary material associated with this article can be found, in In total, higher values statistically significant of HCV/OCI-RNA were achieved in RBCs samples of the patients in the AVP group comparatively to NAVP (p<0.01) and CPP (p < 0.05) patients' groups by ddPCR, although for the other blood samples no statistically significant differences were shown. the online version, at doi:10. 1016/j.virusres.2023.199104.