(P-BC-22) Detection of Oropouche Virus RNA in Blood Donor Minipools During the 2023–2024 Epidemic in Manaus, Brazil: qPCR Assay Validation, Yield, Viral Load and Implications for Transfusion Safety

Background/Case Studies: The Oropouche virus (OROV), an arbovirus endemic to the Amazon basin, reemerged in Brazil in 2023, triggering the largest recorded epidemic with >23,000 confirmed clinical cases. Genomic analyses identified a novel reassortant lineage, with enhanced replication and partial immune escape. Recent reports confirmed vertical transmission, causing fetal demise and congenital anomalies. Although no transfusion-transmitted cases have been reported to date, the OROV viremic period, potential asymptomatic infections and recent spread to urban areas raise blood safety concerns. No systematic studies of OROV viremia in blood donations have been conducted.

Study

Design/Methods: We adapted a reverse transcription quantitative PCR (RT-qPCR) assay targeting the OROV S segment, originally developed by Naveca et al. (2017). We performed in-house validation to confirm analytic performance and ensure assay suitability for pooled donor plasma screening. We then characterized OROV viremia rates in minipools (MPs, 18 plasma samples/pool) from blood donations collected by Hemoam, Manaus, Amazonas, during the recent outbreak.

Results/Findings: Amplification efficiency using in vitro transcribed RNA (IVT) diluted in water was 100.8%. Linearity was confirmed using IVT spiked into negative plasma with lysis buffer (R²=1.0) and cultured OROV isolate dilutions (strain 240023; R²=0.998). Probit analysis with 20 replicates/dilution established a 95% limit of detection (LOD) of 167 copies/mL (95% CI: 86.9–304) and 50% LOD of 19.2 copies/mL (95% CI: 6.3–33.9). The lower limit of quantification (LLOQ), defined by a 25% coefficient of variation cutoff, was 1000 copies/mL. Clinical specificity based on testing 44 healthy donor samples from the United States was 100%. We screened 545 Manaus donor MPs, comprised of 9,810 donations, from November 2023 to April 2024, that had previously tested negative for chikungunya virus, dengue virus (except one), and Zika virus. We detected 42 OROV-reactive MPs (7.7%); 18 of these yielded viral load values above the LLOQ. Most positives (31/42) occurred in January 2024, aligning with peak transmission in the area (Figure).

Conclusions: The assay enables sensitive and quantitative detection of OROV in donor MP samples. Molecular data will be analyzed in combination with OROV serology results of 1001 individual donations from November 2023 and June 2024 to derive estimates of seasonal incidence and the length of MP nucleic acid detection period. Correlation of these data with clinical case reports will allow estimation of disease penetrance. The apparent spread of this reassorted OROV to other areas in the Americas raises questions on the risk of recipient exposure in donated blood products.