Paper-based detection of HIV-1 drug resistance using isothermal amplification and an oligonucleotide ligation assay

Abstract

Regular HIV-1 viral load monitoring is the standard of care to assess antiretroviral therapy effectiveness in resource-rich settings. Persistently elevated viral loads indicate virologic failure (VF), which warrants HIV drug resistance testing (HIVDRT) to allow individualized regimen switches. However, in settings lacking access to HIVDRT, clinical decisions are often made based on symptoms, leading to unnecessary therapy switches and increased costs of care. This work presents a proof-of-concept assay to detect M184V, the most common drug resistance mutation after first-line antiretroviral therapy failure, in a paper format. The first step isothermally amplifies a section of HIV-1 reverse transcriptase containing M184V using a recombinase polymerase amplification (RPA) assay. Then, an oligonucleotide ligation assay (OLA) is used to selectively label the mutant and wild type amplified sequences. Finally, a lateral flow enzyme-linked immunosorbent assay (ELISA) differentiates between OLA-labeled products with or without M184V. Our method shows 100% specificity and 100% sensitivity when tested with samples that contained 200 copies of mutant DNA and 800 copies of wild type DNA prior to amplification. When integrated with sample preparation, this method may detect HIV-1 drug resistance at a low cost and at a rural hospital laboratory.

Rapid molecular detection of macrolide resistance

“…In contrast to LAMP, genomic detection of antimicrobial resistance by RPA is still in its infancy and more progress has been made toward identifying single nucleotide polymorphisms that convey drug resistance. In one study, an HIV drug resistance allele was detected by RPA combined with an oligonucleotide ligation assay [20]. Another study identified multidrug resistant tuberculosis sequence variants using a nested RPA approach [28].…”

“…Eukaryotic pathogens detected with RPA include the blood-fluke Schistosoma japonicum [15] and the diarrheal protozoan pathogens Giardia, Cryptosporidium, and Entamoeba [17,18]. Viral pathogens detected by RPA include HIV [19,20], Chikungunya virus (CHIKV) [14], Rift Valley Fever virus [21,22], Middle East respiratory syndrome coronavirus [23], foot-and-mouth disease virus (FMDV) [24], Bovine Coronavirus [25], and Crimean-Congo Haemorrhagic fever Virus (CCHFV) [26]. Bacterial pathogens detected by RPA include Mycoplasma tuberculosis [27,28], Neisseria gonorrhoeae, Salmonella enterica, and methicillin-resistant Staphylococcus aureus (MRSA) [29], Chlamydia trachomatis [30], Francisella tularensis [31], Group B Streptococci [32], Orientia tsutsugamushi (scrub typhus), and Rickettsia typhi (murine typhus) [16].…”

“…In diagnostic applications RPA has been shown to be highly specific and thus resistant to false positives (Type I errors). In several cases 100% specificity was shown [14][15][16]20]. Because of the health risks of erroneous detection and treatment, high specificity is an important characteristic of diagnostic assays.…”

Rapid screening of UPB1 gene variations by high resolution melting curve analysis

“…At present, mutation detection methods have been relatively established, such as TaqMan probe-based PCR and the molecular beacon method, which have higher requirements for probe design ( 25 ). Besides, molecular hybridization-based methods, such as dynamic allele-specific hybridization and oligonucleotide ligation assays, which also involve expensive sequence-specific probes, increases the cost of detection ( 18 , 19 , 26 , 27 ). Other PCR-combined molecular methods are also available, such as single-strand conformation polymorphism and denaturing gradient gel electrophoresis ( 28 , 29 ).…”

Global Inequities in Precision Medicine and Molecular Cancer Research

“…New, more portable approaches to molecular testing are being developed, which may be useful in resource-limited settings. Paper-based oligonucleotide based ligation assays, originally developed to detect HIV genotypes and monitor resistance, could be adapted for use in cancer therapy ( 94 , 95 ). Paper-based immunochromatography assays, popularised for detection of HIV, could also be adapted for the detection of cancer specific ligands at the point of care.…”