B-144 Performance of IgM ELISA and RT-PCR for Oropouche Virus diagnosis: defining the diagnostic window

Abstract

The Oropouche virus (OROV), primarily transmitted by the bite of infected Culicoides paraensis midges, has emerged as a significant public health concern in South America due to the substantial increase in the number of cases across several countries since late 2023, including both autochthonous and imported transmission, reflecting the virus's spreading potential. Given that symptoms overlap with other arboviruses, such as dengue and chikungunya, laboratory testing is crucial for differential diagnosis. The diagnostic window varies throughout the clinical course of the disease: RT-PCR detection is most effective during the early stages, whereas IgM serology becomes more valuable in later phases. Aiming for a better understanding of disease progression, this study evaluated the relationship between days post-symptom onset (DPSO), the anti-IgM ratio (ELISA), and RT-PCR results for OROV to provide insights for diagnosis based on clinical evolution. Serum samples (n=180) from patients in Espirito Santo State (Brazil) presenting symptoms consistent with arboviruses were tested. Analysis included an in-house RT-PCR targeting the S gene of OROV and by an indirect IgM ELISA (EUROIMMUN), with results expressed as ratio (<0.8: negative; 0.8 to <1.1: indeterminate; =1.1: positive). The DPSO ranged from 2 to 31 days. Data were stratified as follows: <5 days (n=2), =5–7 days (n=135), =8–10 days (n=25) and >10 days (n=18). Overall, 50.6% (91/180) of samples tested positive for OROV in at least one assay, of these, 14.3% (13/91) were double-positive, 46.2% (42/91) were RT-PCR positive only, and 39.6% (36/91) were IgM positive only. Total positivity was 27.2% (49/180) for RT-PCR and 30.6% (55/180) for IgM ELISA, with 6.7% (12/180) yielding borderline results. Sampling was concentrated in the 5–7 DPSO window, representing 75% (135/180) of the cohort. A shift in the detection sensitivity of each method was observed as DPSO increased. In the <5 days group, both available samples (n=2) were detected by RT-PCR but were IgM non-reactive. In the 5–7 days window, both methods showed similar positivity: RT-PCR at 24.4% (33/135) and IgM ELISA at 25.9% (35/135), however, only 3 samples were double-positive, indicating that both tests are complementary in this window. Beyond 8 days, a notable increase in IgM positivity relative to RT-PCR was observed: for the =8–10 and >10 days strata, IgM reactivity was 48% (12/25) and 44.4% (8/18), respectively, while RT-PCR positivity was 40% (10/25) and 22.2% (4/18). The higher proportion of IgM-reactive samples suggests greater clinical utility for serology in the later stages of the disease. The findings reinforce the temporal complementarity between RT-PCR and IgM ELISA for OROV diagnosis. A significant proportion of RT-PCR positive cases remain IgM negative for up to 7 days, and vice versa, while IgM reactivity increases substantially from 8 days onwards. In clinical scenarios with a longer duration of symptoms, IgM serology provides critical diagnostic value as viral load decreases and RT-PCR sensitivity declines.