Can NextGenerationJanuary 14, 2019
Advances in nucleic acid sequencing have been taking place at a great pace since 2005, resulting in several novel Next Generation Sequencing (NGS) systems. Current NGS technology has a three-step approach, in which a DNA library is prepared, enriched and sequenced or identified. Adding bioinformatics tools to this data gives an immense power to detect and identify microbiomes which are hitherto not known. This enables identification of new viruses or mutations using metagenomics approaches. Continuous and dynamic development of NGS technology, coupled with metagenomics, will change the scope of the application of this technology, and enable identifying intraspecies changes within a given biospecimen. Present day diagnostics use a Sanger sequencing-based method for molecular detection, which is not very sensitive.
For example, in case of Transmitted drug-resistance mutations (TDRM) of HIV-1 infection, a comparative study of NGS and Sanger sequencing (Roy Moscona et al., 2017) was performed. It is well known that TDRM frequency may vary in the viral pool. It was noted that one could observe more non-synonymous amino acid substitutions and TDRM using NGS compared to the Sanger method. In the study, an overall TDRM prevalence of 8.8% was identified via Sanger method out of a reported prevalence of 10.1% in treatment-naïve individuals with NRTI as the most affected drug class. However, NGS was able to identify 31.3% of the patients, including those with very low HIV-1 viral load — even below 5%. This suggests that NGS can truly identify viral populations with high genetic diversity and can evaluate at an early stage patients who may develop resistance in the long run. Another study (Casadellà et al., 2016) using an NGS platform found a K65R prevalence of nearly 70% in subjects developing virological failure in first-line antiretroviral therapy (ART) containing TDF (tenofovir), which was missed by Sanger sequencing.
ART is provided in low- and middle-income countries (LMIC) as a public health approach and policy. This leads to HIV drug resistance. However, no regular testing for the drug resistance is done. The present drug resistance testing is primarily for surveying to inform national and regional ART. NGS can become the best-suited technology platform if it is used in centralized laboratories to reduce the cost. This approach can enable large population coverage and identify non-responders or patients who are yet to develop drug resistance.
NGS is likely to soon become a very important cornerstone technology for improved capabilities in diagnosing HIV drug resistance. The clinical value, prevalence of certain mutations and genetic barriers in drug resistance can be best understood and evaluated using NGS. Ultrasensitive genotyping has been proven to improve ART outcome predictions in treatment naïve subjects who are about to start on nevirapine or efavirenz and CCR5 antagonists. It has become very clear that if we are to tackle HIV global pandemic, the question of making NGS accessible to LMICs must be resolved. In the best case scenario — in which global HIV-1 eradication is seen as a possibility — reducing costs in library preparation and bioinformatic analysis will be a good place to start.
Writer is medical scientist and former director of SGRF, Bangalore