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LAMP Assay Developed to Diagnose High HBV DNA Levels

By LabMedica International staff writers
Posted on 05 May 2021
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Image: The AMPLIX real-time polymerase chain reaction system (Photo courtesy of Biosynex)
Image: The AMPLIX real-time polymerase chain reaction system (Photo courtesy of Biosynex)
Worldwide, 257 million people are chronically infected with hepatitis B virus (HBV) and 887,000 annually die from cirrhosis or liver cancer. Since more than 95% of HBV-infected people live in low-income and middle-income countries (LMICs) and only 12-25% of infected people are eligible for anti-HBV therapy.

Loop-mediated isothermal amplification (LAMP) assay is a nucleic acid test (NAT) using DNA polymerase with high auto-cycling strand displacement activity and six specially designed primers. LAMP has the following characteristics allowing its use as a rapid, reliable and inexpensive point-of-care test in LMICs with a high amplification efficiency enabling rapid detection of nucleic acids.

A large team of medical scientists associated with the Pasteur Institute (Paris, France) designed Pan-genotypic primer sets on conserved HBV gene regions. Accuracy of LAMP to identify highly viremic patients was evaluated in 400 and 550 HBV-infected people in France and Senegal, respectively. Analytical validation was performed using real-time turbidimetric LAMP (Loopamp LA-500, Eiken Chemical, Japan). Viral loads were quantified using an AMPLIX real-time PCR (Biosynex, Illkirch-Graffenstaden France).

The team reported that their primers successfully detected eight major HBV genotypes/sub-genotypes (A1/2/3/B/C/D/E/F) with a detection limit ranging between 40-400 IU/mL. In France, the area under the receiver operating characteristic curve (AUROC), sensitivity and specificity of bead-based extraction and real-time turbidimetric LAMP were 0.95, 91.1% and 86.0%, respectively, to diagnose HBV DNA ≥20,000 IU/mL; and 0.98, 98.0% and 94.6% for ≥200,000 IU/mL. The performance did not vary by viral genotypes. In Senegal, using a field-adapted method, reagent-free boil-and-spin extraction and inexpensive end-point fluorescence detection, the AUROC, sensitivity and specificity were 0.95, 98.7% and 91.5%, respectively, to diagnose HBV DNA ≥200,000 IU/mL. The assay was not adapted to discriminate low-level viremia.

The authors concluded that they had developed a simple, rapid (60 minutes), and inexpensive (USD 8/assay) alternative to PCR to diagnose high viremia ≥200,000 IU/mL. HBV-LAMP may contribute to eliminating HBV mother-to-child transmission by identifying high-risk pregnant women eligible for antiviral prophylaxis in resource-limited countries. The study was published on April 7, 2021 in the journal Clinical Microbiology and Infection.

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