Development of a multiassay algorithm (MAA) to identify recent HIV infection in newly diagnosed individuals in Indonesia

Abstract

Ongoing HIV transmission is a public health priority in Indonesia. We developed a new multiassay algorithm (MAA) to identify recent HIV infection. The MAA is a sequential decision tree based on multiple biomarkers, starting with CD4⁺ T cells >200/μL, followed by plasma viral load (pVL) > 1,000 copies/ml, avidity index (AI) < 0 · 7, and pol ambiguity <0 · 47%. Plasma from 140 HIV-infected adults from 19 hospitals across Indonesia (January 2018 - June 2020) was studied, consisting of a training set (N = 60) of longstanding infection (>12-month) and a test set (N = 80) of newly diagnosed (≤1-month) antiretroviral (ARV) drug naive individuals. Ten of eighty (12 · 5%) newly diagnosed individuals were classified as recent infections. Drug resistance mutations (DRMs) against reverse transcriptase inhibitors were identified in two individuals: one infected with HIV subtype C (K219Q, V179T) and the other with CRF01_AE (V179D). Ongoing HIV transmission, including infections with DRMs, is substantial in Indonesia.

Keywords: Computer science; Health sciences; Medicine.

Graphical abstract

Figure 1

Flowchart of participant and specimen selection Long-standing individuals made a training set to develop the serology AI EIA that has never been applied to the Indonesian population. Newly diagnosed individuals were the testing set of this study, where recent infection was identified using MAA.

Figure 2

Distribution of AI values in long-standing and newly diagnosed individuals In individuals with long-standing infection (N = 60), 53 (88 · 33%) have AI ≥0 · 7, and were classified as not recent, while seven (11 · 67%) have AI <0 · 7 that is the characteristics of recent infection. Among newly diagnosed individuals (N = 80), 51 (63 · 75%) have AI ≥0 · 7, suggesting long-standing infection that was just recently diagnosed, while 29 (36 · 25%) have AI <0 · 7. The newly diagnosed individuals having AI <0 · 7 were classified as potentially recent and subjected to ambiguity analysis.

Figure 3

DNA sequencing strategy of the HIV pol gene The sequencing strategy covers the HIV-1 pol gene that consists of protease (PR) amino acids 1–99, reverse transcriptase (RT) amino acids 1–560, and integrase (IN) amino acids 1–288. NFLG cDNA was synthesized in two separate reactions, each using the GSPs Pan-HIV 1F or Pan-HIV 4R. Pre-sequencing amplification was done using both cDNAs as templates in nested amplification. The target region was initially amplified in two large fragments, using primer set 1 that covers HXB2 1031–5308 (4278-bp) and primer set 2 that covers HXB2 2030–4604 (2575-bp). Amplicon 1 was re-amplified into 4 overlapping fragments: F0 (1031–2682), F1 (2030–3817), F2 (2519–4604), and F3 (2984–5308) whereas amplicon 2 was re-amplified into 3 overlapping fragments: FA (2519–3817), FB (2030–3410), and FC (2984–4604). All fragments were sequenced using several overlapping primers, 8 primers each for F0 – F3 and 6 primers each for FA – FC (Table S3).

Figure 4

MAA classifying recent infection The MAA considers recently infected individuals in a hierarchical selection starting from CD4⁺ T cells >200 per μL, pVL >1,000 copies/ml, AI <0 · 7, and HIV pol ambiguity <0 · 47%. In 80 newly diagnosed, ARV naive, adult PLWH, 45 (56 · 25%) had CD4⁺ T cells ≤200 per μL and 35 (43 · 75%) had CD4⁺ T cells >200 per μL. Almost all individuals with CD4⁺ T cells >200 per μL had pVL >1,000 copies/ml (N = 34). Further differentiation based on the AI values resulted in a larger proportion had the long-standing AI ≥0 · 7 (N = 21). The remaining 13 individuals having CD4⁺ T cells >200 per μL and pVL >1,000 copies/ml and AI <0 · 7 were differentiated based on the ambiguity of HIV pol sequence. In the end, 10 individuals presented concordant evidence of recent HIV infection in the MAA (CD4⁺ T cells >200 per μL, pVL >1,000 copies/ml, AI <0 · 7, and HIV pol ambiguity <0 · 47%).