Sequence ambiguity determined from routine pol sequencing is a reliable tool for real-time surveillance of HIV incidence trends.

Identifying individuals recently infected with HIV has been of great significance for close monitoring of HIV epidemic dynamics. Low HIV sequence ambiguity (SA) has been described as a promising marker of recent infection in previous studies. This study explores the utility of SA defined as a proportion of ambiguous nucleotides detected in baseline pol sequences as a tool for routine real-time surveillance of HIV incidence trends at a national level. A total of 353 partial HIV-1 pol sequences obtained from persons diagnosed with HIV infection in Slovenia from 2000 to 2012 were studied, and SA was reported as a percentage of ambiguous base calls. Patients were characterized as recently infected by examining anti-HIV serological patterns and/or using commercial HIV-1 incidence assays (BED and/or LAg-Avidity assay). A mean SA of 0.29%, 0.14%, and 0.19% was observed for infections classified as recent by BED, LAg, or anti-HIV serological results, respectively. Welch's t-test showed a significant difference in the SA of recent versus long-standing infections (p < 0.001). CD4+ T-cell counts ≤250 cells/mm significantly correlated with higher SA (p < 0.001), whereas the homo/bisexual transmission route significantly correlated with lower SA (p = 0.005). When the LAg-assay was used as an indicator of recent infection, a receiver operating characteristic curve with the largest area under the curve (0.896) was observed for SA (sensitivity and specificity of 79%), indicating the best correlation of the data. A reliable estimation of the trends of HIV incident infection could be inferred from measuring SA irrespective of the cutoff used; however, in Slovenia it seems that lower cutoffs are more appropriate. Our data suggest that SA could be used as a real-time surveillance tool for close monitoring of HIV incidence trends, especially in countries where baseline HIV resistance genotyping is performed routinely, rendering this approach cost-effective.