Protocol for the identification of recent HIV infection in newly diagnosed individuals

Recent HIV infection often goes undetected, which complicates incidence estimation, a key metric for understanding HIV spread. We present a protocol to estimate infection recency in newly diagnosed individuals using a multi-assay algorithm (MAA) integrating immunological and virological biomarkers of HIV disease progression. We describe steps for CD4⁺ T cell enumeration, viral load quantification, antibody avidity testing, RNA extraction, nested PCR of HIV-1 pol, Sanger sequencing, and sequence ambiguity quantification. This approach enables recency determination when seroconversion dates are unavailable. For complete details on the use and execution of this protocol, please refer to Wulan et al.¹.