Dados do Trabalho

Título

DEVELOPMENT OF RAPID MOLECULAR DIAGNOSIS FOR HEPATITIS B VIRUS INFECTION TO IMPROVE ACCESS TO DIAGNOSIS IN LOW RESOURCE SETTINGS

Introdução

There are 257 million people who are chronically infected with the hepatitis B virus (HBV) and 887,000 die annually from cirrhosis or liver cancer. Polymerase chain reaction is considered the gold standard method for detecting HBV DNA in serum samples. Despite the high morbidity of HBV infection, many individuals go undiagnosed, especially in areas with limited resources due to the high cost of assays and equipment, sophisticated laboratory infrastructure, and trained personnel to perform an accurate and reliable diagnosis. In this way, Loop-Mediated Isothermal Amplification (LAMP) can be executed in a few minutes, facilitating access to diagnosis in low-resource areas with lower cost and easier to perform compared to PCR.

Objetivo(s)

This study aims to develop LAMP assay for detection of HBV DNA in serum samples.

Material e Métodos

A total of 61 individuals with HBV chronic infection and 20 healthy subjects gave serum samples for this study. Serum samples were tested using commercial real time PCR (Abbott RealTime HBV Viral Load) and genotyped using direct sequencing. Primers were designed to amplify the HBV pre-surface/surface antigen region gene. LAMP was performed at 60°C for 45 min and detection was made using agarose gel electrophoresis.

Resultados e Conclusão

All HBV chronic cases tested positive by using real time PCR (mean±SD viral load of 3.38±2.05 log copies/mL). Using LAMP, HBV DNA was detected in 44 of 61 (72.1%) samples showing a range of detection from 2.78 to 9.00 HBV log copies/mL with nonspecific reaction, demonstrating 100% specificity. Discordant samples among LAMP and real time PCR presented mean viral load of 2.21 HBV log copies/mL. LAMP could amplify samples from the following genotypes: A (56 %), D (48 %), E (24%) and F (24%). LAMP technique presented high specificity and moderate sensitivity and could detect samples from different HBV genotypes. In this way, LAMP is a promising tool to be used in environments with limited resources, specifically in individuals showing high HBV viral load.

Palavras-chave

Rapid molecular diagnosis, LAMP, hepatitis B, new technologies.