The quantitative detection of HCV RNA has been pivotal for initiating treatment and monitoring RNA levels in patients undergoing therapy. Numerous commercial qPCR kits are available for the detection and quantification of HCV RNA [8,9,10,11,12,13]. Among these assays, selecting the most accurate and reliable kits is essential for ensuring effective clinical management. This study evaluated the performance of the DiaRD HCV RTqPCR kit, which was designed to quantitate HCV RNA in a broad dynamic range. We showed that this novel HCV RNA quantification kit exhibited good performance with a sensitivity of 98.8%, a specificity of > 99.9%, and an accuracy of 99.6%. Furthermore, correlation analysis performed on 85 HCV RNA positive samples delivered high concordance with the expected HCV RNA values for the DiaRD HCV RTqPCR and the comparator kits, Artus HCV QS-RGQ kit version 1 (QIAGEN GmbH, Germany) and NeuMoDx HCV Quant assay (Qiagen). Moreover, the observed discrepancy >  ± 0.5 log10 (ranging from 0.59 to 1,2 log10) in only a few plasmas supported similarity between the results of the novel kit and the comparator kits. The results of the kit were reproducible with a CV of ≤ 11.73% for intra-assay, ≤ 17.16% for inter-assay, and ≤ 15.38% for inter-load repeats. The novel kit had the ability to give positive results with a clinical sample having HCV RNA load of 20.8 IU/ml with a 95% certainty. Primers and probe used in the novel kit were highly specific, which gave positive results with all major HCV genotypes without any false-positive reaction with other pathogens.

In HCV RNA positive clinical samples, linear regression analysis showed a strong correlation between the HCV RNA levels measured with novel kit and those of the comparator tests. The obtained R2 values of 0.915 indicate that more than 91% of the measurements obtained with the novel kit are comparable to those of the comparator kits. These results indicated that the measurement outcomes of the novel DiaRD-HCV kit are highly consistent, and HCV RNA load in clinical samples can be accurately measured. Besides, the slope of the linear regression equation (y = 0.902x + 0395) demonstrates that the measurement values of the DiaRD-HCV kit are nearly like those of the comparators, allowing researchers and clinical practitioners to compare test results. In agreement with our results, previous studies also found high concordance between qPCR methods. A recent study showed that a fully automated microfluidic RT-qPCR system was highly correlated with the Roche cobas AmpliPrep/cobas TaqMan HCV Test, version 2.0 (r2 = 0.949 [14]. Another study reported a correlation (R2 = 0.828, p = 0.001) of HCV RNA levels in clinical samples detected by the Artus HCV RT PCR and RTA RT-PCR assays [12]. In a study, quantitation performance of the Abbott Real Time HCV assay and the Roche Cobas TaqMan HCV assay was compared, and it was reported that HCV RNA loads detected with the two assays were highly correlated (R2 = 0.94) [13]. A recent study performed on 39 HCV RNA positive samples found a high correlation (R2 = 0.97) between the Aptima HCV Quant Dx,and Roche Cobas 6800 platform (C6800) systems [9]. These data indicate that even if there are small differences between commercial kits, most of them able to quantitate HCV RNA loads within comparable ranges.

To show the agreement between the quantitative results of DiaRD HCV RTqPCR and comparator kits, Bland–Altman analysis was performed by drawing a scatter plot of the differences against the means of measurements obtained from two tests. Accordingly, while the DiaRD HCV RTqPCR kit assay was measured slightly higher, the mean difference of 0.12 log10 IU/ml was within the acceptable clinical threshold of ± 0.5 log10 IU/mL, with most measurements falling within the 95% confidence interval. Like our result, in a previous study, the Bland–Altman analysis performed on the results of the fully automated microfluidic RT-qPCR system and the Roche cobas AmpliPrep/cobas TaqMan HCV Test, version 2.0 showed that the bias between the two methods were distributed almost evenly in the detection range [14]. In another study, the mean difference between RTA-RT PCR and Artus RG RT-PCR assays in QCMD reference samples was found to be 0.4 log IU/ml [12]. The other study performed to compare diagnostic performance of a new developed one-step real-time PCR assay with Cobas/Taqman HCV Test v2.0 (Roche) found a significant correlation on virus load results of two systems (p = 0.012) [15]. Our findings align with previous studies that reported high concordance between different qPCR systems for HCV RNA quantification.

Before using any qPCR kit for routine diagnosis, it is very important to know the limit of detection/analytical sensitivity of the assay to be sure for its detection ability the low-level viremia to understand those at risk of being mis-diagnosed [16]. According to the manufacturer’s instructions, the limit of detection of the NeuMoDx HCV Quant assay and artus HCV QS RGQ kit version 1 was 8 IU/ml and 21 IU/ml, respectively. A previous study showed that the analytical sensitivity of the HCV specific NASBA assay was 100–150 IU/ml [10]. In another study, the limit of detection of a microfluidic RT-qPCR system was reported as approximately 12 IU/ml [14]. In a recent study, it was reported that a laboratory optimized quantitative RT-PCR method had a limit of detection of 42.6 IU/mL (95% CI, 32.84 to 67.76 IU/mL) [17]. The current study confirmed that The DiaRD HCV RTqPCR kit can detect HCV RNA ≥ 20.8 IU/ml in the samples with a 95% certainty. This finding suggests that the DiaRD HCV kit can reliably detect HCV RNA even in samples with low viral loads, making it suitable for both diagnosis and treatment monitoring.

The reason for the discrepancy between PCR assays may be due to the difference in sample volumes used in RNA extraction: i.e.,200 μl for DiaRD HCV RTqPCR, 500 μl for the artus-HCV RTqPCR systems, and 700 μl for the NeuMoDx system [17]. Also, the elution volume can result in a difference in the RNA quantification. RNA extracted from a high sample volume and resuspended in low-elution buffer increases the detection rate in low-viral-load samples [18]. This argument is supported by one sample not detected in the DiaRD HCV RTqPCR kit, being positive with a 66 IU/ml HCV RNA with the NeuMoDx HCV system. The other reasons may also be the different target size and regions used for detection of viral genome [19]. It was stated that using a short target increased the sensitivity of PCR method [17]. The DiaRD HCV RTqPCR kit targets to amplify the 105-base pairs region within the 5'untranslated region (5'UTR) of the HCV genome, while the Artus HCV QS-RGQ kit uses primers for specific amplification of a 240 bp region of the HCV genome. The other reason might be the difference in analytic sensitivities of the kits [15]. The 95% LoD of the DiaRD HCV RNA RTqPCR assay was 20.8 IU/ml, which is equal to the LoD value (21 IU/ml) of Artus HCV QS-RGQ kit, however it is higher than that of the NeuMoDx HCV system (LoD value of 8 IU/ml). The negative result of the novel kit on the sample having the 66 IU/ml HCV RNA load with the NeuMoDx HCV system might be resulted from its higher LoD value than that of comparator kit. It is most probable this sample might be also negative with the Artus HCV QS-RGQ kit. Unfortunately, we were not able to test this sample with the Artus HCV QS-RGQ kit to clarify this argument. The discrepancy between PCR systems can be related to viral loads in clinical samples. In agreement with this statement, a previous study showed that Roche C6800 system yielded mostly lower viral loads than the Cobas Ampliprep/Cobas TaqMan v2.0 and the Abbott RealTime assay towards to low viral loads, and higher values in the upper viral loads [20]. In our study the difference on the viral loads was not restricted to low or high RNA levels. Of the 12 clinical samples showing differences of > 0.5 log10, six had RNA loads > 4 log10. In the discrepancy samples, the novel kit detected slightly high viral loads in nine samples, of which five had RNA load of > 4 log10. These factors responsible for discrepancy emphasize the importance of assay-specific parameters when interpreting results.

Several studies have shown that HCV genotypes did not significantly affect performance of qPCR kits and did not make a difference in the quantitation performance of the kits [13, 20]. A recent study indicated that quantification of HCV RNA loads by the VERIS HCV assay, and the Abbott RealTime HCV assay showed well correlated for all HCV genotypes, except genotype 4 [21]. Parallel to these results we could not find a significant difference in the quantification of the HCV genotype 1a, 1b, 2, 3a, 5, and 6 in SeraCare’s reference materials. These genotypes could be quantitated with low differences ranging from 0.01 to 0.26 log10 IU/ml. However, in genotype 4 having viral load of 4.16E + 04, a −0.61 log10 difference was observed. Low differences observed in quantification of HCV genotypes between the DiaRD HCV RTqPVR kit and reference values support the robustness of the assay across diverse genotypes. Although we have not been able to detect HCV genotypes in clinical samples to expand accuracy of our results on genotypes circulating in study population, recent studies on the Turkish population have shown that genotype 1 is predominant, accounting up to 95% of HCV strains, followed by genotype 3 [22, 23].

The intra-assay, inter-assay, and inter-load assay reproducibility studies of the novel kit detected standard deviation (SD) values ranging from 6.20 × 101 to 8.54 × 103, and from 2.30 × 101 to 1.43 × 104, and 1.13 × 102 to 5.69 × 102 IU/ml values, respectively. The CV values of this kit were ≤ 11.73% for intra-assay, ≤ 17.16% for inter-assay, and ≤ 15.38% for inter-load repeats. A study using a fully automated microfluidic RT-qPCR system demonstrated that the assay had a reproducibility of SD ≤ 0.16 log10 IU/ml [17]. Another study reported a reproducibility with 2.52% and 1.33% CVs for inter- and intra-assays, respectively for a novel real-time RT-PCR assay [24]. In current study, the mean intra- and inter-assay CV for NeuMoDx HCV Quant Assay was reported as < 5% [8]. According to the instructions of artus® HCV RG RT-PCR Kit, CV values for intra-assay and inter-assay were 6.34% and 9.98%, respectively. According to results of six HCV RNA levels tested in three replicates per level in 12 runs on four days by using the AmpliPrep/COBAS® TaqMan® HCV Qualitative Test, v2.0 kit, the CV values were reported between 9 and 54%. It was indicated that CV values ​​below 15% are generally considered acceptable [25]. The coefficient variation values ​​of the DiaRD HCV RTqPCR kit agreed with those of existing commercial kits used in routine diagnosis of HCV infection.

By using results of the comparator kits, the diagnostic sensitivity and specificity of the novel kit were found to be ≥ 99.9%. In agreement with our results, a recent study performed with loop-mediated reverse transcription isothermal amplification demonstrated a sensitivity of 91.5% and specificity of 100% [26]. According to kit’s instruction, the sensitivity and specificity of the COBAS® AmpliPrep/COBAS® TaqMan® HCV Qualitative Test, v2.0 are 100% and 100%, respectively (https://www.aphl.org/programs/infectious_disease/Documents/CAPCTM%20HCV%20v2%20US-IVD%20PI%20rev4%20(Dx%20claim).PDF). These values are 100% and 98.6% for the artus® HCV QS RT-PCR Kit (https://www.qiagen.com/gb/products/diagnostics-and-clinical-research/infectious-disease/artus-hcv-pcr-kits). Both sensitivity and specificity of Abbott RealTime HCV kit are 100% (https://extranet.who.int/prequal/sites/default/files/whopr_files/PQDx_0450-027-00_AbbottRealTimeHCV_PR_v3.0.pdf). DiaRD HCV TRqPCR kit showed good performance in detection and quantification of HCV RNA levels, with similar sensitivity and specificity values as compared to another commercial methods. Detecting all HCV genotypes without any false positive results with the tested pathogens provided additional data regard to accuracy of the novel kit.

Despite its strengths, this study has some limitations. The performance characteristics of the DiaRD kit were not evaluated on a larger cohort of clinical samples with diverse HCV genotypes, nor was it tested for its ability to monitor viral load changes in patients undergoing treatment. Future studies should focus on these aspects to enhance the generalizability of our findings and validate the kit’s performance in a broader clinical context. The other limitation is that due to financial limitation, we could not test all samples being compared across all kits included in the comparison. It’s possible that a sample showing the positive result with NeuMoDX HCV kit but negative with the novel kit might be also negative with Artus HCV QS-RGQ kit, other comparator kit. So, the negative result obtained by the novel kit may not be false negative.

In conclusion, the high correlation between quantitative results of the DiaRD-HCV RNA RTqPCR kit and the comparator kits, along with its high sensitivity, specificity, and accuracy indicates the diagnostic reliability of this novel kit. Besides, its analytical sensitivity supports the ability of the test to detect low viral loads. The DiaRD kit’s ability to detect all major HCV genotypes [1,2,3,4,5,6] further highlights its clinical utility. These results indicate that the DiaRD HCV RTqPCR kit has the potential to become an integral tool for diagnosis and quantitation of HCV RNA in patients.