Development of quantitative multiplex RT-qPCR one step assay for detection of hepatitis delta virus

Hepatitis Delta is a disease caused by exposure to hepatitis B (HBV) and hepatitis D (HDV) viruses, usually with a more severe clinical outcome when compared to an HBV monoinfection. To date, the real prevalence of HDV infection is underestimated and detection methods are poorly available, especially in more endemic regions. Therefore, a one-step RT-qPCR method for quantification of HDV-RNA was developed. Biological samples were selected between 2017 and 2023 from patients at the Ambulatório Especializado em Hepatites Virais of the Centro de Pesquisa em Medicina Tropical de Rondônia and Serviço de Assistência Especializada and underwent the test developed by this study and a second quantitative RT-qPCR assay. The slope of the initial quantitative assay was − 3.321 with an efficiency of 100.04% and amplification factor equal to 2. Analysis of the repeatability data revealed a Limit of Quantification of 5 copies/reaction and Limit of Detection (95%) of 2.83 copies per reaction. In the diagnostic sensitivity tests, there was an accuracy of 97.37% when compared to the reference test. This assay proved to be highly efficient and reproducible, making it a valuable tool to monitor hepatitis Delta patients and assess the risk of disease progression, as well as the effectiveness of treatment.

Diagnostic sensitivity and specificity. Blood samples were collected from 266 patients with chronic infection, positive for anti-HDV, being followed up for Delta hepatitis, of which 16.54% (44/266) were identified as having liver disease and following treatment. Of this total, 64.6% (172/266) were men, aged between 24 and 77 years (mean 45 years; standard deviation (SD) ± 10. 23). The samples were tested using the assay developed, where 22.93% (61/266) had a quantifiable viral load, in a range of 2.96 copies per reaction (2.17 Log10 copies/  Table S4). A total of 11.47% (7/61) of positive samples were negative for the reference test 14 , which corresponds to 2.63% (7/266) of samples tested. When compared to the reference test, the development assay showed 100% (95% CI: 93. 40 Figure S2). The divergent samples had a viral load of less than 7 copies per reaction (2.96-6.07 copies/reaction), which may explain the disagreement in the assay results and low viral load. All samples negative for HDV and positive for other viruses (DENV, SARS-CoV, and HBV monoinfected, N = 26) showed negative results when subjected to the current test.

Discussion
Hepatitis delta is a public health problem in endemic areas in South America. This is a neglected disease that affects vulnerable populations, such as indigenous and riverside dwellers in the Amazon basin 5 . Table 1. Repeatability and Reproducibility Assays. The reproducibility and repeatability assays were performed in three runs on alternate days with dilutions in technical octuplicates, with coefficient of variation (CV%) less than 10% between runs.
In this study, an assay was developed to quantify HDV RNA viral load different with high sensitivity and efficiency. The one step RT-qPCR technique developed in the present study showed good efficiency and reproducibility, which is significant for greater precision in quantification from a standard curve.
The methodology used was based on procedures described in the literature and respects quantitative international standards for molecular diagnosis, where the alignment of parameters for carrying out RT-qPCR assays is essential to reduce the risk of false positives and inconclusive results, which decreases assay reliability 16,17 . The assay meets the established criteria for validating a quantitative qPCR, such as efficiency, linear dynamic range, Limit of Detection (LOD) and accuracy, which ensures its robustness 16 .
The efficiency estimated from the slope showed values close to 100% and an amplification factor equal to 2, in accordance with guidelines for standardization of tests that consider a high-performance test with values between 90 and 110% 16,18 . Likewise, the linear dynamic range was determined with 5 different concentrations (Log10), with a minimum of 3 being recommended 16,19,20 , in addition to intra-assay (repeatability) and interassay (reproducibility) precision expressed by Standard Deviation (SD) measurements of replicates on a standard curve 16,21,22 .
The LOD was defined as the lowest value tested with 5% of failed reactions, which demonstrated a limit of 142 copies per mL, being similar to the 130 copies/mL obtained in our previous study; however in the current assay, in addition to the addition of an Endogenous control for monitoring reaction quality, the assay allows Reverse Transcription and qPCR to be performed in a single reaction 23 . In comparison, other assays for quantification of HDV-RNA showed a limit of detection that ranged from 15 to 750 copies/mL [24][25][26] .
During the experiments on patient samples, an improvement in the quantification of HDV-RNA was observed by applying the heat shock step before the Reverse Transcription step. The same result was observed in a study that evaluated the effect of shock in serially diluted clinical samples 27 . This could be explained by the nature of the HDV genome, which has a high G/C ratio and which makes internal base pairing ~ 70% 2 .
In this study, we chose to use a recombinant plasmid as the assay quantification standard that presented a consensus sequence of the 8 HDV genotypes, bearing in mind that the Amazon region presents a predominance of Genotype 3 10,28,29 , and the standard established by the WHO for HDV quantification is derived from genotype 1 30 . Likewise, the primers used in the standardization were designed in order to detect a conserved region for the 8 virus genotypes, guaranteeing the sensitivity and specificity of the test. It is important to point out that despite its practical application, the pattern constructed with a partial HDV genome has its limitations because it is not a real biological sample, however, we used the plasmid diluted in a matrix of nucleic acids extracted from a negative sample to HDV to mimic a biological sample.
The results of 7 samples that disagreed between the current test and reference test can be explained by the borderline values for quantification, however, the comparison was limited only to qualitative results because Additionally, most of patients in this study have received therapy for hepatitis Delta, which justifies the fact that despite the positive serology for HDV, not all of them had a quantifiable viral load, as observed in both assays performed.
When it comes to HDV detection, diagnosis can be made both by detecting anti-HDV antibodies and by searching for direct markers, such as the HDV antigen, and by detecting the circulating viral genome 15,31 . One of the main limitations of diagnosing HDV using serological method is the failure to elucidate the current infection, being limited to a marker of contact with the virus, which is different from what occurs for HBV, where it is possible to determine the different stages of that disease 32 . In this case, the use of a quantitative assay can help in understanding the different stages of HDV infection.
Serological diagnosis is not widely applied in Brazil, which makes it difficult to know the real prevalence of HDV in this country. It is important to raise awareness and efforts by health authorities and population for the importance of serological diagnosis. According to a recommendation of a guideline to Diagnosis of Viral Hepatitis in Brazil, hepatitis Delta should be investigated in individuals who present reactive results in immunoassays for HBsAg and who reside or have been in endemic areas for this condition 33 .
According to WHO, there is still a limitation in the availability of HDV diagnosis and absence of direct HDV RNA detection methods, which are normally used to monitor the response to antiviral therapy 34 . As with HBV and HIV, the standardization of laboratory diagnosis by Real-Time PCR allowed significant advances in the clinical protocol and therapeutic guidelines for adequate treatment and control, with more specific approaches aimed at patients 35 . The expansion of diagnostic coverage for HDV in Brazil would enable the elaboration of guidelines for specific treatment and the diagnostic algorithm (for identification in suspected cases), reducing the risk of evolution, with remission of acute and chronic liver activities (decrease of manifestations and necroinflammatory activities, cirrhosis and hepatocellular carcinoma) 36 .
In this early decade, researchers still addressed issues to establishing a satisfactory treatment for those patients infected with HDV chronic, even though the current therapy with Peg-IFN (pegylated interferon) is dated with approximately 35 years of medical availability 32,37 . The existing obstacles in relation to the effectiveness of the treatment for HDV are associated with the direct dependence on the prescription of therapies aimed at the treatment of HBV in infected individuals. Thus, the therapeutic approach is conditioned to circulating HBsAg levels, based on international guidelines, which direct the management of medications in individuals with HDV only when, in clinical follow-up, they present a viral load for HBV > 2,000 IU/mL 38,39 . In this case, the application of a quantitative RT-qPCR assay for HDV detection will allow identifying, quantifying, and monitoring viral replication during the course of the disease in patients.
In addition to an accurate serological and quantitative molecular diagnosis for HDV, it is necessary to raise the awareness of health agencies and governments on this issue, with special attention to countries with a high number of confirmed cases. Vaccine Programs to HBV, but also mapping HDV endemic areas, tracking cases and providing properly treatment to HDV cases could be useful to prevent future transmission.
In conclusion, Tthis current assay has demonstrated high efficiency and reproducibility, making it a valuable tool to monitor HDV carriers and assess disease progression as well as treatment efficacy. In addition, this assay may allow studies to link viral load levels and persistence of chronic HDV, what brings higher risk of developing cirrhosis and hepatocellular carcinoma.  Figure S3). The plasmid concentration in ng/μL was determined by fluorimetry using the Qubit 4 dsDNA High Sensitivity kit (ThermoFisher Scientific®, Massachusetts, USA) and the initial copy number of 2.12 × 10 8 was calculated based on the recombinant DNA size and molecular weight, according to the previously described method 41 , based on the following Eq. (1):
Quantitative one-step multiplex RT-qPCR assay. To quantify HDV-RNA, primers and probes were designed to detect the Ribozyme region, which is highly conserved 42 , as seen in Supplementary Figure S3. To disrupt secondary structures of HDV genome, genetic material extracted from biological samples was incubated at 95 °C for 5 min followed by ice for 1 min. The multiplex reaction was standardized using 5 µL of TaqMan Fast Virus 1-Step Master Mix (Applied Biosystems®, California, USA), 10 µL of each curve dilution or genetic material from biological sample, 5 µL of oligomix containing 300 nM of each primer and 100 nM for the HDV probe, and 500 nM of each primer and 125 nM of probe for detection of Endogenous Control ( Assay performance analysis. Reproducibility and repeatability were measured through 3 tests performed on consecutive days by different operators. The points with 1 × 10 5 ; 1 × 10 4 ; 1 × 10 3 ; 1 × 10 2 ; 1 × 10 1 ; 5; 2.5 and 1.25 copies/reaction were used in technical octuplicates for each day. Analytical sensitivity was determined using repeatability data, which served as the basis for measuring the Limit of Detection (LOD95%).
Diagnostic sensitivity and specificity. To determine the diagnostic sensitivity, the biological samples were submitted to test developed to HDV RNA quantification. All tested samples were subjected to a second commercial quantitative RT-qPCR test (RealStar® HDV RT-PCR Kit 1.0; Altona Diagnostics, Hamburg, Germany) as a reference 14 and compared to the data obtained. Positive samples for other viruses and negative for HDV (DENV, SARS-CoV, and monoinfected HBV) were tested using the assay developed to estimate diagnostic specificity.
Statistical analysis. Descriptive analyses were represented through frequencies, central tendency, and dispersion. The Standard Curve was performed using R v4.2.1 44 software and to calculate the Limit of Detection (1) copy number = plasmid concentration ng/µL plasmid bp number + insert bp number × 650 ×avogadro ′ s constant 6, 02 x 10 23 www.nature.com/scientificreports/ (LOD95%), the analytical sensitivity data were subjected to binomial regression analysis using the Probit statistical model. The diagnostic sensitivity and specificity results were used to estimate the values of sensitivity, specificity, Positive Predictive Value, Negative Predictive Value, Kappa Agreement Index and construction of the ROC curve, using reference assay 14 .

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.