EVALUATION OF A POINT OF CARE IMMUNOFILTRATION ASSAY PROTOTYPE FOR DETECTION OF GROUP A ROTAVIRUS IN HUMAN SAMPLES

PERI IBAÑEZ ES; MANDILE MG; FLORES C; KIKOT P; SILVESTRE D; TOMÁS FARIÑA J; ARGÜELLES M; TEMPRANA CF; GLIKMANN G; GRASSELLI M; CASTELLO AA

Bali

Simposio; 14th International Rotavirus Symposium; 2023

Comité de organizadores del 14th International Rotavirus Symposium

Background and aimsRotaviruses continue to be an important cause of morbidity and mortality in young children. Although licensed vaccines have proven to be effective in countries of medium to high socioeconomic status, their efficacy and effectiveness in low-income regions and poor socio-sanitary conditions show a much lower performance. The availability of Point-of-Care test (POCT) would be extremely useful for monitoring the circulation of atypical variants potentially escaping immune protection induced by vaccines. Emergence of these variants is typical within deprived populations located in semi-rural areas where the probability of infections with high viral load inocula and mixed content in human or human-animal strains is maximized due to poor sanitary conditions and frequent contact with breeding animals. The goal of this study is to evaluate the performance of a lab-in-a-syringe-format immunofiltration assay (IFA) for detection of group A Rotavirus (RVA) using low-cost supplies. Lab-in-a-syringe tests have shown to be more sensitive than lateral flow assays and more inexpensive to produce.MethodsWe used track-etched PET membranes porous surface as the reaction space carried out through passive antigen-antibody contact. We evaluated different chemical etching times for the production of nanochannels and observed and measured their shape and size by SEM. We used a dot-blot assay to determinate the concentration of gold nanoparticles (AuNPs) and the preparation of antibodies and their concentration to be used for the test as well as some of the assay parameters. We used RVA positive and negative fecal samples from patients previously tested by ELISA. Then, the dot-blot assay was replaced by a syringe and a filter holder for its evaluation as a potential Lab-in-a-syringe Point of Care methodology format (figure 1). Finally, to corroborate that RVA detection was carried out within the membrane nanochannels we treated the tested membranes with pyrrole and iron chloride to form polypyrrole nanotubes and subsequently visualize them by SEM.ResultsWe established an antibody concentration of 0.2 mg/ml to be immobilized on the membrane and a sample volume of 0.3 ml and it was possible to reduce the reaction time to 5’ (figure 1). The performance comparison between the IFA and the reference ELISA showed a 96% of sensitivity and a 98% of specificity. We were able to corroborate that the RVA detection occurs within the nanochannels of the track-etched membrane by SEM observation of AuNPs within the pyrrole nanotubes.ConclusionsWe developed a prototype of IFA based in the active solution permeation into the channels of the membrane in which capture antibodies are uniformly accumulated. This allows an efficient contact with the target molecules and provides high sensitivity even for a short period of time. Their unique morphology and a large available surface area and the efficient utilization of the channel space will lead to a promising material for POCT. The syringe format prototype seems promising, so it is necessary to continue working to ensure the stability of the test components and the introduction of an internal control and then evaluate sensitivity and specificity with a larger number of samples.