Nanotechnology: A reality for diagnosis of HCV infectious disease.

Hepatitis C virus (HCV) is the primary etiologic agent of liver cirrhosis or hepatocellular carcinoma. HCV elevated infection rates are mostly due to the lack of an accurate and accessible screening and diagnosis, especially in low- and middle-income countries. Conventional HCV diagnostic algorithm consists of a serological test followed by a nucleic acid test. This sequence of tests is time consuming and not affordable for low-resource settings. Nanotechnology have introduced new promising tests for the diagnose of infectious diseases. Based on the employment of nanoparticles and other nanomaterials which lead to highly sensitive and specific nanoscale tests, most of them target pathogen genome. Implementation of nanoscale tests, which are affordable, portable and easy to use by non-specialized personal, would improve HCV diagnosis algorithm. In this review, we have summed up the current emerging nanotechnology tools, which will improve actual screening and treatment programs, and help to reach HCV elimination proposal.Financial support was provided by the Community of Madrid , call for grants for the completion of Industrial Ph.D. to VB and RM ( IND2017/BMD-7683 ).S

Development of a novel and rapid molecular diagnostic system for Hepatitis C virus (HCV) detection.

Hepatitis C virus (HCV) is an enveloped, single-stranded positive sense RNA [ssRNA(+)] virus which causes cirrhosis, hepatocellular carcinoma and liver failure in infected patients [1]. The Global Hepatitis Report posted in 2017, indicates that around 71 million people were living with HCV in 2015 [2]. Moreover, the most recent data collected in 2019 by WHO was able to estimate that 58 million people live with chronic hepatitis C infection and about 1.5 million new infections occur per year. Due to the increasing number of infections, one of the recommendations postulated in the 2022 guidelines ("Updated recommendations on HCV simplified service delivery and HCV diagnostics: policy brief") was to achieve more efficient and simplified hepatitis diagnosis [3]. The diagnosis of an active HCV infection requieres the detection of HCV RNA, and nowadays it is performed by the gold standard real-time reverse transcription polymerase chain reaction (RT-qPCR). Nevertheless, it involves specific laboratory facilities, well-trained personnel and high-cost equipment, maintenance and reagents [4]. Consequently, this project was born on the need of developing a new diagnostic system for direct HCV molecular detection based on the RT-LAMP (reverse transcription loop-mediated isothermal amplification) technique that could be implemented in resource-limited settings. This strategy is focused on the nucleic acid-based amplification method using from four to six primers under isothermal conditions to amplify specifically and effectively the target sequence [4]. The aim of the project is to validate the system and integrate it in a future point-of-care (POC) diagnostic device

903 Protein Saver cards: the best alternative for dried blood spot storage at room temperature for HCV RNA

Hepatitis C virus (HCV) infection remains a global health problem, detected only in the early stages by molecular tests. Molecular tests detect HCV RNA, which is very prone to degradation by ribonucleases, reason why blood samples must be transported and stored at − 20 °C, or even − 70 °C for long-term storage. Flinders Technology Associates (FTA) cards are a useful sampling collecting device for dry blood spot (DBS) storage, especially for low and middle-income countries (LMIC). In this study, we analyzed viral HCV RNA integrity for long-term storage at room temperature compared to − 20 °C using two diferent types of cards for DBS: FTA Classic and 903 Protein Saver cards. For this purpose, DBS were prepared on these cards using blood or plasma samples from HCV infected patients, and samples were analysed by conventional RT-PCR. Our results showed that 903 Protein Saver cards are the best and cheapest alternative for DBS storage at room temperature. In these conditions, we found that HCV RNA integrity lasted for up to 9 months

Desarrollo de un sistema “Point of Care” de diagnóstico molecular para la detección del Virus de la Hepatitis C mediante nanosondas-ADN

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 20-07-2022Esta Tesis tiene embargado el acceso al texto completo hasta el 31-05-202

A Novel, Rapid Diagnostic Molecular Method for Sars-Cov-2 Detection by Nanoprobes

Early diagnosis of active viral infections is crucial to prevent silent transmission among the population, as we have seen during the present COVID-19 pandemic. Single stranded RNA viruses, including SARS-CoV-2, are currently detected using molecular methods, such as the gold standard qRT-PCR. These methods require expensive equipment and specialized training, which limits its use as point-of-care (POC) diagnostic systems. We have developed a novel, fast and simple diagnostic method, based in the combination of Isothermal Loop Amplification (LAMP) [1], [2] and oligonucleotide-functionalized gold nanoparticles (AuNPs). If viral RNA is amplified and recognized by de DNA probe, a change in gold nanoparticles aggregation state initiates, leading to a wavelength change in the region of the visible spectrum, evident by naked eye. The developed system has been designed by BioAssays SL to detect SARS-CoV-2. After successful results detecting synthetic targets, we have pre-validated our novel diagnostic system with a 364 sample panel from SARS-CoV-2 positive patients (83 positive samples and 281 negative samples). We analyzed two different primer sets, targeting Gen-E and Gen-N respectively, and we determined that the Gen-N primer set reliably detected SARS-CoV-2 RNA with an RT-qPCR cycle threshold (CT) number of up to 30, with a 98% sensitivity and 100% specificity compared to commercial qRT-PCR diagnostic kits. These results showed that RT-LAMP is sensitive enough to detect viral RNA within 30 min and a detection limit of 150 copies per mL when using these primers. To allow a colorimetric visualization of the results, we coupled LAMP amplification to AuNPs detection [3], [4]. DNA-AuNPs (nanoprobes) were able to detect LAMP results in a saline buffer in 15 minutes, leading to aggregation in the absence of RT-LAMP amplification. Then, nanoprobes remained stable in saline buffer when they specifically recognized SARS-CoV-2 RT-LAMP product, and the red colour of the solution was indicative of a positive result. All positive samples amplified by RT-LAMP were successfully detected by nanoprobes. In conclusion, our LAMP detection system coupled to nanoprobe visualization is sensitive enough to detect viral RNA within less than 60 min by the naked eye, with 98% sensitivity and 100% specificity, overcoming sensitivity limitations of previously described nanoprobe systems [5]. Due to the potential threat of re-emerging we have also designed a LAMP-nanoprobe detection system for Hepatitis C virus and we are currently starting pre-validation phases too. Upon in vivo validation, a marketable diagnostic kit will be developed that can be implemented in the health system as a routine method for assay. The estimated cost for the diagnostic kit is 5€ per assay. The simplicity of this technology allows its ready transfer and optimization for to the detection of other viral diseases by RNA or ssDNA viruses such as measles virus, Zika, Dengue, HIV-1, or even animal viruses, such as Avian Metapneumovirus (aMPV).Depto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu

Epidemic history and baseline resistance to NS5A-specific direct acting drugs of hepatitis C virus in Spain.

Hepatitis C virus (HCV) infection remains a global health problem. Previously, the prevalence of NS5A resistance-associated substitutions (RASs) to elbasvir, a new direct-acting antiviral (DAA) against the NS5A viral protein was assessed by our group before its introduction into clinical use in Spain. However, the origin, epidemic history, transmission dynamics, diversity and baseline RASs to NS5A direct-acting agents of HCV-GT1a in Spain remain unknown. A nationwide cross-sectional survey of individuals chronically-infected with HCV-G1a and DAAs-naïve was performed. HCV population sequencing, phylogenetic analysis and Bayesian methods were used. GT1a clade II was more prevalent than clade I (82.3% vs. 17.7%; P < 0.001) and older (estimated origin in 1912 vs. 1952). Clade II epidemic is currently declining whereas clade I epidemic has reached equilibrium. A total of 58 single RASs were identified, which account for the moderate level (10%) of baseline resistance observed. When considering the regional data, marked differences were observed, with thirteen regions showing an intermediate level (5-15%) and one a high level (20%) of resistance. Current HCV-GT1a epidemic in Spain is driven by clade I which seem to have different dissemination routes relative to clade II. A moderate level of baseline RASs to NS5A-DAAs with marked differences among regions was observed. Close surveillance of response to treatment with DAAs will be important.The Instituto de Salud Carlos III (ISCIII) provided financial support by the Grant (CP13/00098, PI15CIII/00031 and PI18CIII/00020 to VB). CP is financed by national funds via FCT through Norma Transitória—DL57/2016/CP1376/CT0004.S

Epidemic history and baseline resistance to NS5A-specific direct acting drugs of hepatitis C virus in Spain

Hepatitis C virus (HCV) infection remains a global health problem. Previously, the prevalence of NS5A resistance-associated substitutions (RASs) to elbasvir, a new direct-acting antiviral (DAA) against the NS5A viral protein was assessed by our group before its introduction into clinical use in Spain. However, the origin, epidemic history, transmission dynamics, diversity and baseline RASs to NS5A direct-acting agents of HCV-GT1a in Spain remain unknown. A nationwide cross-sectional survey of individuals chronically-infected with HCV-G1a and DAAs-naïve was performed. HCV population sequencing, phylogenetic analysis and Bayesian methods were used. GT1a clade II was more prevalent than clade I (82.3% vs. 17.7%; P < 0.001) and older (estimated origin in 1912 vs. 1952). Clade II epidemic is currently declining whereas clade I epidemic has reached equilibrium. A total of 58 single RASs were identified, which account for the moderate level (10%) of baseline resistance observed. When considering the regional data, marked differences were observed, with thirteen regions showing an intermediate level (5–15%) and one a high level (20%) of resistance. Current HCV-GT1a epidemic in Spain is driven by clade I which seem to have different dissemination routes relative to clade II. A moderate level of baseline RASs to NS5A-DAAs with marked differences among regions was observed. Close surveillance of response to treatment with DAAs will be important.The authors thank patients and the Contributing members of the Spanish Group of Chronic Viral Hepatitis (SI1 Appendix) that have participated in this epidemiological survey and the Multidisciplinary Group of Viral Coinfection HIV/Hepatitis (COVIHEP). The Instituto de Salud Carlos III (ISCIII) provided financial support by the Grant (CP13/00098, PI15CIII/00031 and PI18CIII/00020 to VB). CP is financed by national funds via FCT through Norma Transitória—DL57/2016/CP1376/CT0004.info:eu-repo/semantics/publishedVersio

Hepatitis C Virus Influences HIV-1 Viral Splicing in Coinfected Patients

Coinfection with hepatitis C virus (HCV) influences HIV reservoir size. However, it is unknown whether this coinfection also induces a higher provirus transcription. Viral transcription is promoted by synergy between cellular factors such as NF-κB and the viral regulator Tat. The impact of HCV coinfection on HIV provirus transcription was analyzed in resting (r)CD4 T+ cells (CD3+CD4+CD25-CD69-HLADR-) and rCD4 T cells-depleted PBMCs (rCD4 T- PBMCs) from a multicenter cross-sectional study of 115 cART-treated HIV patients: 42 HIV+/HCV+ coinfected individuals (HIV+/HCV+), 34 HIV+ patients with HCV spontaneous clearance (HIV+/HCV−) and 39 HIV patients (HIV+). Viral transcription was assessed in total RNA through the quantification of unspliced, single spliced, and multiple spliced viral mRNAs by qPCR. Linear correlations between viral reservoir size and viral splicing were determined. A 3-fold increase of multiple spliced transcripts in rCD4 T+ cells of HIV+/HCV+ patients was found compared to HIV+ individuals (p < 0.05). As Tat is synthesized by multiple splicing, the levels of Tat were also quantified in these patients. Significant differences in single and multiple spliced transcripts were also observed in rCD4 T- PBMCs. Levels of multiple spliced mRNAs were increased in rCD4 T+ cells isolated from HIV+/HCV+ subjects, which could indicate a higher Tat activity in these cells despite their resting state.Financial support was provided by the Instituto de Salud Carlos III to VB and AFR (PI15CIII/00031 and PI18CIII/00020), by the Spanish Ministry of Economy and Competitiveness to MC (SAF2016-78480-R), and the SPANISH AIDS Research Network RD16CIII/0002/0001 and RD16CIII/0002/0002-ISCIII—FEDER. A.F.R. is supported by the Miguel Servet programme from Fondo de Investigación Sanitaria (ISCIII) (CP14/CIII/00010).S

Low-level HIV-1 viremia affects T-cell activation and senescence in long-term treated adults in the INSTI era

Background Around 10% of people with HIV (PWH) exhibit a low-level viremia (LLV) under antiretroviral therapy (ART). However, its origin and clinical significance are largely unknown, particularly at viremias between 50 and 200 copies/mL and under modern ART based on integrase strand transfer inhibitors (INSTIs). Our aim was to characterize their poor immune response against HIV in comparison to individuals with suppressed viremia (SV) and non-HIV controls (NHC). Methods Transversal observational study in 81 matched participants: 27 PWH with LLV, 27 PWH with SV, and 27 NHC. Activation (CD25, HLA-DR, and CD38) and senescence [CD57, PD1, and HAVCR2 (TIM3)] were characterized in peripheral T-cell subsets by spectral flow cytometry. 45 soluble biomarkers of systemic inflammation were evaluated by immunoassays. Differences in cell frequencies and plasma biomarkers among groups were evaluated by a generalized additive model for location, scale, and shape (GAMLSS) and generalized linear model (GLM) respectively, adjusted by age, sex at birth, and ART regimen. Results The median age was 53 years and 77.8% were male. Compared to NHC, PWH showed a lower CD4+/CD8+ ratio and increased activation, senescence, and inflammation, highlighting IL-13 in LLV. In addition, LLV showed a downtrend in the frequency of CD8+ naive and effector memory (EM) type 1 compared to SV, along with higher activation and senescence in CD4+ and CD8+ EM and terminally differentiated effector memory RA+ (TEMRA) subpopulations. No significant differences in systemic inflammation were observed between PWH groups. Conclusion LLV between 50 and 200 copies/mL leads to reduced cytotoxic activity and T-cell dysfunction that could affect cytokine production, being unable to control and eliminate infected cells. The increase in senescence markers suggests a progressive loss of immunological memory and a reduction in the proliferative capacity of immune cells. This accelerated immune aging could lead to an increased risk of developing future comorbidities. These findings strongly advocate for heightened surveillance of these PWH to promptly identify potential future complications.Instituto de Salud Carlos IIICentro de Investigación Biomédica en Red de Enfermedades InfecciosasBioAssaysDepto. de Genética, Fisiología y MicrobiologíaFac. de Ciencias BiológicasTRUEpu

Dynamics of HIV Reservoir and HIV-1 Viral Splicing in HCV-Exposed Individuals after Elimination with DAAs or Spontaneous Clearance

Background: Although human immunodeficiency virus type 1 (HIV-1) reservoir size is very stable under antiretroviral therapy (ART), individuals exposed to the Hepatitis C virus (HCV) (chronically coinfected and spontaneous clarifiers) show an increase in HIV reservoir size and in spliced viral RNA, which could indicate that the viral protein regulator Tat is being more actively synthesized and, thus, could lead to a higher yield of new HIV. However, it is still unknown whether the effect of HCV elimination with direct-acting antivirals (DAAs) could modify the HIV reservoir and splicing. Methods: This longitudinal study (48 weeks&rsquo; follow-up after sustained virological response) involves 22 HIV+-monoinfected individuals, 17 HIV+/HCV- spontaneous clarifiers, and 24 HIV+/HCV+ chronically infected subjects who eliminated HCV with DAAs (all of them aviremic, viral load &lt; 50). Viral-spliced RNA transcripts and proviral DNA copies were quantified by qPCR. Paired samples were analyzed using a mixed generalized linear model. Results: A decrease in HIV proviral DNA was observed in HIV+/HCV- subjects, but no significant differences were found for the other study groups. An increased production of multiple spliced transcripts was found in HIV+ and HIV+/HCV+ individuals. Conclusions: We conclude that elimination of HCV by DAAs was unable to revert the consequences derived from chronic HCV infection for the reservoir size and viral splicing, which could indicate an increased risk of rapid HIV-reservoir reactivation. Moreover, spontaneous clarifiers showed a significant decrease in the HIV reservoir, likely due to an enhanced immune response in these individuals