Labor-intensive techniques (e.g.: RT-qPCR) impede early detection of YFV in NHP, humans, and mosquitoes, delaying the implementation of YF surveillance programs due to the need for specialized equipment and skilled technicians19. In this study, RT-LAMP assays using three primers targeting different molecular regions (NS1, NS5, and E) streamline YFV detection, reducing processing time compared to RT-qPCR, and overcoming limitations in early YFV detection through both molecular and serological methods20,21.
Initial experiments revealed that the NS1 primer for YFV13 failed to amplify South American strains (Accession numbers: OP508651.1, OP508679.1, OP508678.1, OP508680.1, OP508683.1, OP508684.1, OP508652.1) from a recent outbreak in Southern Brazil (2019 to 2021). This failure was due to genetic polymorphisms at the primer binding site, as it was originally designed based on older strains from Bolivia, Colombia, Ecuador, French Guiana, Panama, Peru, Trinidad, Venezuela, the vaccine strain, and Brazil that circulated between 1980 and 200213. Indeed, Meagher et al (2018)22 noted that the NS1 region is not completely conserved across YFV lineages from Africa, the 17D vaccine strain, and South America. To tackle this, in this study new degenerate primers targeting conserved NS5 and E regions of the YFV genome were designed to broaden coverage across multiple virus strains, enhancing test robustness and sensitivity22. These, alongside the NS1 primer13, were employed in RT-LAMP reactions for specific YFV RNA detection. Therefore, it is recommended to periodically review the YFV diagnostic primers used in both RT-qPCR or RT-LAMP assays to identify mutations and reduce the risk of false negatives.
Several molecular diagnostic protocols for YFV have been proposed13,19,20,21,23, yet none have been evaluated using field-collected samples from NHP tissues. Despite Nunes et al.’s (2011)21 findings, which demonstrated higher sensitivity in detecting YFV in experimentally infected hamster liver samples (by PCR and RT-qPCR techniques), the RT-LAMP assay showed potential in detecting YFV across all NHP samples, even in tissues such as lung, heart, and kidney, which are commonly known to have low YFV viral loads2.
The limit of detection identified in this study is found to be equivalent to 12 PFU/mL (Fig. 4a-b), a result like that demonstrated by Nunes et al13 of 19 PFU/mL using RT-LAMP, and equivalent to those observed using RT-qPCR (9 PFU/mL)21. Following Astari et al24 findings, which interpret orange results as indicative of amplification, the assay's detection limit might be even lower than 12 PFU/mL (Fig. 4c).
Also, our study found no cross-reaction with other arboviruses, including DENV1-4 and ZIKV, indicating the specificity of the RT-LAMP assay for YFV detection, mitigating a common potential limitation of Flavivirus diagnostic25. Even when other flaviviruses were present in a pooled experiment (DENV1-4 + ZIKV + YFV), the RT-LAMP assay showed high sensitivity, with no interference in YFV detection. Additionally, in the absence of YFV in a flavivirus pool, no amplification occurred, confirming the specificity of the primers designed for YFV detection.
The RT-LAMP assay developed for NHPs is expected to detect YFV in humans and mosquitoes, as the primers were designed based on prevalent strains in these groups. It exhibits 100% specificity and sensitivity (Fig. 2, Table 1), comparable to the RT-qPCR technique21, making it a reliable, cost-effective, and user-friendly alternative for YFV molecular diagnosis. It addresses critical gaps present in other protocols20,21, offering easily interpretable results without sophisticated equipment. Our goal is to improve surveillance of NHP epizootics, humans, and mosquitoes, to enable a prompt response to prevent human YFV outbreaks.