Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance
JOURNAL OF MOLECULAR DIAGNOSTICS
2019; 21 (4): 580?92
Molecular epidemiology of hepatitis C virus in Cambodia during 2016-2017.
2019; 9 (1): 7314
Multiplex Solid-Phase Melt Curve Analysis for the Point-of-Care Detection of HIV-1 Drug Resistance.
The Journal of molecular diagnostics : JMD
In Cambodia, little epidemiological data of hepatitis C virus (HCV) is available. All previous studies were limited to only small or specific populations. In the present study, we performed a characterization of HCV genetic diversity based on demography, clinical data, and phylogenetic analysis of HCV non-structural 5B (NS5B) sequences belonging to a large cohort of patients (n=3,133) coming from majority part of Cambodia between September 2016 and December 2017. The phylogenetic analysis revealed that HCV genotype 1 and 6 were the most predominant and sharing equal proportions (46%). The remaining genotypes were genotype 2 (4.3%) and unclassified variants (3.6%). Among genotype 1, subtype 1b was the most prevalent subtype accounting for 94%. Within genotype 6, we observed a high degree of diversity and the most common viral subtypes were 6e (44%) and 6r (23%). This characteristic points to the longstanding history of HCV in Cambodia. Geographic specificity of viral genotype was not observed. Risks of HCV infection were mainly associated with experience of an invasive medical procedure (64.7%), having partner with HCV (19.5%), and blood transfusion (9.9%). In addition, all of these factors were comparable among different HCV genotypes. All these features define the specificity of HCV epidemiology in Cambodia.
View details for DOI 10.1038/s41598-019-43785-4
View details for PubMedID 31086236
A point-of-care HIV-1 genotypic resistance assay that could be performed during a clinic visit would enable care providers to make informed treatment decisions for patients starting therapy or experiencing virological failure on therapy. The main challenge for such an assay is the genetic variability at and surrounding each drug-resistance mutation (DRM). We analyzed a database of diverse global HIV sequences and used thermodynamic simulations to design an array of surface-bound oligonucleotide probe sets with each set sharing distinct 5' and 3' flanking sequences but having different centrally located nucleotides complementary to six codons at HIV-1 DRM reverse transcriptase position 103: AAA, AAC, AAG, AAT, AGA, and AGC. We then performed in vitro experiments using 80-mer oligonucleotides and PCR-amplified DNA from clinical plasma HIV-1 samples and culture supernatants containing subtype A, B, C, D, CRF01_AE, and CRF02_AG viruses. Multiplexed solid-phase melt-curve analysis discriminated perfectly among each of the six reported reverse transcriptase position 103 codons in both 80-mers and clinical samples. The sensitivity and specificity for detecting targets containing AAC mixed with targets containing AAA were above 98% when AAC was present at a proportion of at or above 10%. Multiplexed solid-phase melt-curve analysis is a promising approach for developing point-of-care assays to distinguish between different codons in genetically variable regions such as those surrounding HIV-1 DRMs.
View details for PubMedID 31026601