Speaker
Description
Australia is aiming to end the transmission of Human Immunodeficiency Virus (HIV) and Hepatitis C Virus (HCV) by 2030. National strategies for both viruses highlight the importance of timely high-quality data to improve public health responses. Such data could be obtained through a nationwide molecular epidemiology surveillance system based on routine long-range viral sequencing. However, for successful implementation a robust, time- and cost-effective method for long-range sequencing must be developed and/or validated.
Primers for two novel tiling PCR assays were designed to amplify HIV or HCV genomes in overlapping segments of ~1kb. Assays were benchmarked using two panels of 90 infected and 6 uninfected samples from archived plasma as per the WHO HIVResNet Method Validation Framework. Panels included several viral subtypes/genotypes over a range of viral loads (HIV: 1,295-1,301,193 copies/mL, HCV: 121-12,813,253 IU/mL). HIV samples were additionally compared with prior diagnostic sequencing to determine the sequence accuracy of the tiling PCR method.
The novel tiling PCR methods generated any viral sequence in 90/90 samples (100%) for both HIV and HCV. Genome coverage was highly variable; however a complete Core-E2 of HCV, or a complete Reverse Transcriptase of HIV, was amplified in 74% and 93% of samples, respectively. The HIV assay had good concordance with prior diagnostic sequencing, with 98% of consensus sequences sharing >99% genetic identity to historical data. For both assays the time from sample to result was <24 hours, at a cost of <$65USD per sample.
These novel tiling PCRs are an attractive option for routine long-range viral sequencing. However, regional sequencing failures may be a result of underlying viral diversity, and sequence dropouts must be monitored in the future. These methods will now undergo pilot deployment in a public health laboratory to determine utility for rollout as part of a nationwide molecular epidemiology surveillance system.
