Fast and Sensitive Real-Time PCR Detection of Major Antiviral-Drug Resistance Mutations in Chronic Hepatitis B Patients by Use of a Predesigned Panel of Locked-Nucleic-Acid TaqMan Probes

Abstract

We developed a novel real-time PCR assay that simultaneously evaluates eleven major nucleos(t)ide antiviral (NA) drug-resistance mutations (mt) in chronic hepatitis B patients (CHB), including L180M, M204I/V, and V207M (lamivudine (LMV) resistance), N/H238A/T (Adefovir (ADF) resistance) which are circulating in Vietnam; T184G/L, S202I, M250V (entecavir (ETV) resistance), and A194T (tenofovir resistance) which have been recently reported in several studies across the globe. We detected drug-resistant mt in HBV samples using our predesigned panel of allele-specific locked-nucleic acid (LNA) probes. Our assay had a high sensitivity of 5% in a low HBV DNA population of ≥5 ×10 3 IU/mL and was validated in a cohort of 130 treatment-naive children and 98 NA-experienced adults with CHB. Single-point mt for LMV- and ADF-resistance were detected in 57.7% and 54.1% of the child and adult samples, respectively, with rtV207M (42.3%, children; 36.7%, adults) and rtN238T/A (15.4%, children; 16.3, adults) being the most frequent mt in these populations. Multiple-point mt including rtL180M-rtM204V- rtN238A and rtL180M-rtM204I were only identified in two children, resulting in LMV-ADF-resistance and reduced ETV susceptibility. In conclusion, this assay accurately identified the mt profile of children (98.4%) and adults (91.2%) with CHB, which is comparable to established methods. This fast and sensitive screening method can be used for the detection of major NA-resistant mt circulating in developing countries, as well as providing a model for the development of similar mt-detection assays, especially for use in non-hospitalised patients who need their results within half a day before starting treatment.

Partial Characterization of Three Bacteriophages Isolated from Aquaculture Hatchery Water and Their Potential in the Biocontrol of Vibrio spp.

“…Furthermore, the 16S rRNA gene of each isolate was sequenced, and an internal control was performed for all possible isolates. The real-time PCR method enabled the discrimination of members of the genus Vibrio in less than 4 h. LNA probes offer significant advantages such as high sensitivity, a wide linear range, a clinically relevant detection limit, ease of design, and improved signal generation [46,47]. LNA probes have the ability to distinguish single-base mutations through amplification curves without the need for complex detection or additional Tm analysis [48].…”

Locked nucleic acid real-time polymerase chain reaction method identifying two polymorphisms of hepatitis B virus genotype C2 infections, rt269L and rt269I

“…LNA-based RT-PCR assays have been widely applied to viral single-nucleotide polymorphism analysis as well as simple viral detection in clinical settings instead of the less sensitive traditional RT-PCR or nested RT-PCR assays prone that are to cross-contamination[ 31 , 32 ]. In particular, it has recently been reported that this method could successfully identify YMDD mutations of HBV from Korean patients with chronic HBV infections[ 30 , 33 ].…”

Trends in Molecular Diagnosis of Nosocomial Pneumonia Classic PCR vs. Point-of-Care PCR: A Narrative Review

“…Besides savings due to faster discharge and less wasteful drug administration, POC-PCR does not require additional qualified personnel. Therefore, nucleic acid analysis with these methods can be carried out by personnel with minimal training, although the advantages of such tests can be influenced by the work technique, especially by delaying testing, which inherently leads to delaying appropriate treatment [32,86,131].…”