Results of clinical trials of a reagent kit for detection of enteroviruses with additional differentiation of enterovirus D68

Background. Human enteroviruses pose a significant public health threat, causing a wide range of clinical manifestations, from mild respiratory illnesses to severe neurological manifestations. Enterovirus D68 (EV-D68) is of particular concern due to its association with outbreaks of severe respiratory and neurological diseases in children worldwide. In the Russian Federation, cases of EV-D68 have been recorded since 2018; however, there are no kits available for differential diagnosis of EV-D68 from other enterovirus infections. Aim. Development and implementation of a reagent kit for the detection of enterovirus RNA with differentiation of EV-D68 in human biological specimens using the RT-PCR method. Materials and Methods. The design of specific oligonucleotides for amplification of enterovirus species A/B/C/D and EV-D68 RNA was carried out using the UGENE software and the NCBI resource. RNA extraction was performed with the «RIBO-prep EV» and «Magno-Sorb-Combo EV» kits. RT-PCR was conducted with fluorescence detection of amplified cDNA for enteroviruses A/B/C/D, EV-D68, and an internal control sample on the HEX, Cy5, and FAM channels. Analytical sensitivity was evaluated using a recombinant MS2 bacteriophage containing an EV-D68 RNA fragment. Specificity was assessed by testing a panel of viral and bacterial strains. Diagnostic performance was evaluated using the «AmpliSens Human enterovirus-FL» kit and VP1 gene sequencing. Results. Analytical sensitivity was evaluated using model samples of human biological material and amounted to 1·10³ GE/mL for nasopharyngeal and oropharyngeal swabs and cerebrospinal fluid, and 5·10³ GE/mL for fecal samples. No cross-reactions were observed with RNA from closely related rhinoviruses, other viruses, or bacteria. To assess diagnostic performance, 175 clinical samples from patients with enterovirus infections (EVI) and 135 samples negative for enterovirus RNA were tested. Results showed complete agreement with those obtained using a PCR kit from another manufacturer; EV-D68 detection was confirmed by VP1 gene sequencing. Diagnostic sensitivity and specificity reached 100% (confidence interval: 86.3—100%; p=0.95). Conclusions. The results of the conducted trials have confirmed that the developed reagent kit exhibits high analytical and diagnostic performance, demonstrating both efficacy and safety. The kit is approved for in vitro diagnostic use and has obtained a Roszdravnadzor registration certificate (No. RU 2024/23935 dated November 7, 2024).