title: Development of a reverse transcription quantitative real-time PCR-based system for rapid detection and quantitation of hepatitis delta virus in the western Amazon region of Brazil.
abstract: The hepatitis delta virus (HDV) is a pathogen that causes a severe and rapidly progressive disease of hepatocytes. The measurement of viral load in the peripheral blood of patients with HDV infections is important for diagnosis, treatment monitoring, and support for follow-up studies of viral replication during the course of the disease. This study reports the development of an assay capable of detecting and quantifying the abundance of HDV particles in serum samples, based on reverse-transcription quantitative PCR (RT-qPCR). Two standards for calibration were produced for determining the viral load of HDV: a cDNA cloned into a linear plasmid and a transcribed RNA. For validating this assay, 140 clinical samples of sera were used, comprising 100 samples from patients who tested positive for anti-HDV and hepatitis B virus surface antigen (HBsAg) by ELISA; 30 samples from blood donors; 5 samples monoinfected with hepatitis B virus (HBV); and 5 samples monoinfected with hepatitis C virus (HCV). The HDV RT-qPCR assay performed better when calibrated using the standard based on HDV cDNA cloned into a linear plasmid, yielding an efficiency of 99.8% and a specificity of 100% in the in vitro assays. This study represents the first HDV RT-qPCR assay developed with clinical samples from Brazil and offers great potential for new clinical efficacy studies of antiviral therapeutics for use in patients with hepatitis delta in the western Amazon region.
authors: Fundação Oswaldo Cruz Rondônia (FIOCRUZ-RO), Laboratório Plataforma Técnica, Brazil; Centro de Pesquisa em Medicina Tropical de Rondônia (CEPEM), Brazil; Universidade Federal de Rondonia (UNIR), Núcleo de Saúde, Departamento de Medicina, Programa de Pós-graduação em Biologia Experimental (PGBIOEXP), Brazil. Electronic address: luan_botelho@hotmail.com.
authors: Luan Felipo
authors: LF
authors: Botelho-Souza
authors: Fundação Oswaldo Cruz Rondônia (FIOCRUZ-RO), Laboratório Plataforma Técnica, Brazil; Centro de Pesquisa em Medicina Tropical de Rondônia (CEPEM), Brazil; Universidade Federal de Rondonia (UNIR), Núcleo de Saúde, Departamento de Medicina, Programa de Pós-graduação em Biologia Experimental (PGBIOEXP), Brazil.
authors: Alcione de Oliveira
authors: Ade O
authors: dos Santos
authors: Centro de Pesquisa em Medicina Tropical de Rondônia (CEPEM), Brazil.
authors: Lourdes Maria
authors: LM
authors: Borzacov
authors: Centro de Pesquisa em Medicina Tropical de Rondônia (CEPEM), Brazil.
authors: Eduardo Resende
authors: ER
authors: Honda
authors: Fundação Oswaldo Cruz Rondônia (FIOCRUZ-RO), Laboratório Plataforma Técnica, Brazil; Centro de Pesquisa em Medicina Tropical de Rondônia (CEPEM), Brazil; Universidade Federal de Rondonia (UNIR), Núcleo de Saúde, Departamento de Medicina, Programa de Pós-graduação em Biologia Experimental (PGBIOEXP), Brazil.
authors: Juan Miguel
authors: JM
authors: Villalobos-Salcedo
authors: Fundação Oswaldo Cruz Rondônia (FIOCRUZ-RO), Laboratório Plataforma Técnica, Brazil; Centro de Pesquisa em Medicina Tropical de Rondônia (CEPEM), Brazil; Universidade Federal de Rondonia (UNIR), Núcleo de Saúde, Departamento de Medicina, Programa de Pós-graduação em Biologia Experimental (PGBIOEXP), Brazil.
authors: Deusilene Souza
authors: DS
authors: Vieira
mesh_terms: D001938:Brazil; D003699:Hepatitis D / Q000175:diagnosis* / Q000821:virology; D003698:Hepatitis Delta Virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load / Q000379:methods*
keywords: HBV; HBsAg; HDV; HDV RNA; Hepatitis delta virus; Hydrolysis probe; Quantitative real-time PCR; RT-qPCR; Viral load; hepatitis B virus; hepatitis B virus surface antigen; hepatitis delta virus; reverse-transcription quantitative PCR