Human pegivirus, Toscana virus and herpesviruses identified in cerebrospinal fluid from adults with unexplained neurologic disease, Spain, 2022–2023

Viral central nervous system (CNS) infections in adults frequently remain unresolved after routine diagnostic testing. We applied probe-based viral metagenomic next-generation sequencing (vmNGS) to cerebrospinal fluid samples from adults with suspected CNS infection and negative conventional diagnostics in a retrospective multicentre study conducted in Spain between 2022 and 2023. Among 40 idiopathic cases, vmNGS detected viral sequences in 6 patients without evidence of coinfection: human pegivirus (HPgV, n =3), Toscana virus (TOSV, n =1), herpes simplex virus type 1 (HSV-1, n =1) and varicella-zoster virus (VZV, n =1). Two HPgV-positive patients were transplant recipients, with neurological disease occurring more than 2 years after transplantation, compatible with possible long-term viral persistence in immunocompromised hosts. TOSV genotype B was identified in a patient residing in central Spain, supporting consideration of TOSV in selected cases of unexplained aseptic meningitis during the vector season, including outside traditionally recognized Mediterranean coastal regions. Furthermore, the failure of syndromic panel testing to detect HSV-1 and VZV highlights the need for complementary diagnostic strategies when clinical suspicion remains high. Overall, the detection of unexpected viral sequences, together with missed clinically actionable infections, supports the use of complementary molecular testing in selected cases of unexplained CNS syndromes when routine diagnostics are negative. These findings highlight the added diagnostic value of vmNGS and provide sequence-level data for future studies of viral diversity and molecular epidemiology in neurological disease.