Rapid Amplicon Nanopore Sequencing (RANS) for the differential diagnosis of Monkeypox virus and other vesicle-forming pathogens

As of July 2022, more than 7000 laboratory-confirmed monkeypox (MPX) cases have been reported worldwide. Until recently MPX was a rare viral disease seldom detected outside Africa. MPX virus (MPXV) belongs to the Orthopoxvirus (OPV) genus and is a genetically close relative of the Variola virus (the causative agent of smallpox). Following the eradication of smallpox, there was a significant decrease in smallpox-related morbidity and in the population immunity to the other OPV-related diseases like MPX. In parallel, there is a need for differential diagnosis between the different OPVs clinical manifestations and diseases with similar symptoms (i.e. Chickenpox, Herpes Simplex). The aim of the current study was to provide a rapid genetic-based diagnostic tool for an accurate and specific identification MPXV and additional related vesicle-forming pathogens. We initially assembled a list of 14 relevant viral pathogens, causing infectious diseases associated with vesicles, prone to be misdiagnosed as MPX. Next, we developed an approach that we termed Rapid Amplicon Nanopore Sequencing (RANS). The RANS approach uses diagnostic regions which harbor high homology in their boundaries and internal diagnostic SNPs that, when sequenced, aid the discrimination of each pathogen within a group. During a multiplex PCR amplification, a dA tail and a 5-phosphonate are simultaneously added, thus making the PCR product ligation ready for Nanopore sequencing. Following rapid sequencing (a few minutes), the reads are compared to a reference database and the closest strain is identified. We first tested our approach using samples of known viruses cultured in cell lines. All the samples were identified correctly and swiftly. Next, we examined a variety of clinical samples from the 2022 MPX outbreak. Our RANS approach identified correctly all the PCR-positive MPXV samples and mapped them to strains that were sequenced at the 2022 outbreak. For the subset of samples that were negative for MPXV by PCR, we got definite results, identifying other vesicle-forming viruses: Human hepersvirus 3, Human hepersvirus 2 and Molluscum contagiosum virus. This work is a proof-of-concept study, demonstrating the potential of the RANS approach for rapid identification of a panel of closely-related pathogens. The simplicity and affordability of our approach makes it easy to implement in any genetics lab. Moreover, other differential diagnostics panels could benefit from the implementation of the RANS approach into their diagnostics pipelines.