Developing High-Throughput HIV Incidence Assay with Pyrosequencing Platform

Human immunodeficiency virus (HIV) incidence is an important measure for monitoring the epidemic and evaluating the efficacy of intervention and prevention trials. This study developed a high-throughput, single-measure incidence assay by implementing a pyrosequencing platform. We devised a signal-masking bioinformatics pipeline, which yielded a process error rate of 5.8 × 10(-4) per base. The pipeline was then applied to analyze 18,434 envelope gene segments (HXB2 7212 to 7601) obtained from 12 incident and 24 chronic patients who had documented HIV-negative and/or -positive tests. The pyrosequencing data were cross-checked by using the single-genome-amplification (SGA) method to independently obtain 302 sequences from 13 patients. Using two genomic biomarkers that probe for the presence of similar sequences, the pyrosequencing platform correctly classified all 12 incident subjects (100% sensitivity) and 23 of 24 chronic subjects (96% specificity). One misclassified subject's chronic infection was correctly classified by conducting the same analysis with SGA data. The biomarkers were statistically associated across the two platforms, suggesting the assay's reproducibility and robustness. Sampling simulations showed that the biomarkers were tolerant of sequencing errors and template resampling, two factors most likely to affect the accuracy of pyrosequencing results. We observed comparable biomarker scores between AIDS and non-AIDS chronic patients (multivariate analysis of variance [MANOVA], P = 0.12), indicating that the stage of HIV disease itself does not affect the classification scheme. The high-throughput genomic HIV incidence marks a significant step toward determining incidence from a single measure in cross-sectional surveys.