Dados do Trabalho

Título

Development of quantitative RT-qPCR one step assay for detection of Hepatitis Delta Virus

Introdução

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, such as the Amazon region, which has high rates of chronic cases of the disease.

Objetivo (s)

The aim of the study was to develop a high sensitivity quantitative one step RT-qPCR assay for the diagnosis and follow-up of patients with Hepatitis Delta.

Material e Métodos

The assay was designed for simultaneous detection of the viral target and an endogenous human control. The quantification curve was constructed from linear regression analysis of 6 serial dilutions using a recombinant plasmid containing a partial region of the HDV ribozyme corresponding to the 8 HDV genotypes. A total of 266 biological samples were collected between 2017-2023 from patients at the Ambulatório Especializado em Hepatites Virais of the Centro de Pesquisa em Medicina Tropical de Rondônia and Serviço de Assistência Especializada do Acre and and submitted to the test developed by this study, in addition to a second quantitative RT-qPCR assay used as a reference test.

Resultados e Conclusão

The slope of the initial quantitative assay was -3.321 with an efficiency of 100.04% and amplification factor equal to 2. 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. 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, which is the lowest viral load value that can be detected using the assay developed. In the diagnostic sensitivity tests, there was an accuracy of 97.44% 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.

Palavras-chave

Hepatitis Delta Virus; Quantitative RT-qPCR; Viral load.

Agradecimentos

This study was funded by Fundação Oswaldo Cruz Rondônia, Instituto Nacional de Ciência e Tecnologia de Epidemiologia da Amazônia Ocidental, and the Fundação Rondônia de Amparo ao Desenvolvimento das Ações Científicas e Tecnológicas e à Pesquisa do Estado de Rondônia (Process: VPPIS-003-FIO-20-2-75).