الفهرس 
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Abstract
Fewer than 20% of those living with HCV infection worldwide have been diagnosed, and the challenge now is to engage, screen, and diagnose everyone in need of treatment.
Nucleic acid testing (NAT) for the detection of HCV RNA remains the gold standard for diagnosing active HCV infections. However, the laboratory setup for performing NAT requires expert technical staff, expensive equipment and reagents, dedicated procedure areas, and availability of serum or plasma samples. Because of these requirements, NAT is not routinely performed in many clinical laboratories.
Gold nanoparticles (AuNPs) have found their way into clinical diagnostics due to their unique optical properties. AuNPs decorated with a specific nucleic acid probe were used for colorimetric detection of HCV RNA. The gold nano-assay is based on using cationic AuNPs to induce the aggregation of AuNPs probes. In the absence of HCV, the cationic AuNPs electrostatically bind to negatively charged AuNPs-DNA probes causing their aggregation and change of the solution color to blue. The presence of HCV nucleic acid protects the probes and prevents their aggregation by cationic AuNPs and the solution color remains red.
This study aimed at evaluation of gold nanoparticle-based assay as a diagnostic method for colorimetric detection of unamplified HCV RNA in clinical samples.
The study was conducted on 75 subjects that were recruited from blood bank unit and outpatient clinic, Hepatology department, National Liver Institute, Menoufiya University.
Subjects were divided into two groups: group I: consisted of 42 apparently healthy blood donors as the negative (disease-free) group, and group II: consisted of 33 patients with Hepatitis C Virus infection (diseased group).
Two types of gold nanoparticles; citrate (negatively charged) and cysteamine (positively charged) were synthesized and characterized at laboratories of centre of genomics, Zewail city of science and technology. Citrate gold nanoparticles were then functionalized with HCV specific probe.
All subjects were submitted to the reference method for diagnosis of active HCV infection, real-time PCR. Then, the gold nano-assay was performed, and results were compared to the gold standard Real-Time PCR method.
Test characteristics of gold nano-assay for discrimination between the studied groups were analysed by ROC curve.
The best cut-off value that was determined by ROC curve to have the highest accuracy (78.8%) was >1.36, it achieved the best discrimination between blood donors and HCV groups with sensitivity of 90.91 %, specificity of 66.67%, The AUC was 0.764 with SE of 0.058 and a highly significant P-value < 0.001. The assay was able to detect viral load as low as 761 IU/ml or 0.95 IU/μL
Clinical usefulness of the assay
HCV testing in clinical settings is important for different clinical purposes and in different disease stages, here we discuss the diagnostic value of the developed gold nano-assay for different clinical purposes.
1- Screening of blood donations
The screening of HCV infection is important for the testing of blood and blood-related products to reduce HCV transmission risk. An international collaborative study state that individual donation NAT with or without anti-HCV testing showed higher efficacy than either minipool NAT (in pools of 8 or 16 donations) or combo assays (HCV Ag/Ab). Residual risk with individual donation NAT and serology screening was estimated at one in 250,000 in Egypt and at one in 10,000,000 in other study regions combined; risk would increase to one in 7300 and one in 312,000, respectively, if NAT had not been performed. (Bruhn et al., 2015)
The main challenge for testing individual donations with NAT is its high cost, so the gold nano-assay can be a good cost-effective alternative for testing individual donations for HCV RNA.
2- Detection of viraemic infection & pre-therapeutic assessment
According to the European Association for the Study of the Liver (EASL) Recommendations on Treatment of Hepatitis C, HCV RNA detection and quantification in serum or plasma should be made as pre-therapeutic assessment by a sensitive assay with a low limit of detection of ≤15 IU/ml. But, In low- and middle-income countries and in specific settings in high-income countries, a qualitative HCV RNA assay with a low limit of detection of ≤1,000 IU/ml can be used if more sensitive quantitative assays are not available and/or not affordable (Pawlotsky et al., 2018).
The developed gold nano-assay is a good qualitative test that was able to detect HCV RNA in serum of patients with viral load of less than 1000 IU/ml, so it can be used for detection of viraemic infection and for pre-therapeutic assessment.
3- Monitoring of treatment efficacy
Considering the high rate of cure, as more potent DAAs become available, the role of confirmation of cure may diminish in the near future (Applegate et al., 2018). Despite that the recent recommendations of EASL on Treatment of Hepatitis C still emphasize that the endpoint of therapy is undetectable HCV RNA in serum or plasma by a sensitive assay (lower limit of detection ≤15 IU/ml) 12 weeks (SVR12) or 24 weeks (SVR24) after the end of treatment. The recommendations also state that qualitative HCV RNA assay with a low limit of detection ≤1,000 IU/ml can also be used as an alternative endpoint of therapy in areas where sensitive HCV RNA assays are not available and/or not affordable (Pawlotsky et al., 2018).
The developed gold nano-assay can also be used for monitoring of treatment efficacy with a technique that’s more simple, less expensive in less equipped laboratories instead of nucleic acid testing.