Performance evaluation of Automated Fluorescent Immunoassay System ROTA and NORO for detection of rotavirus and norovirus: A comparative study of assay performance with RIDASCREEN® Rotavirus and Norovirus

Abstract Background The Automated Fluorescent Immunoassay System ROTA (AFIAS‐Rota) and NORO (AFIAS‐Noro) assays (Boditech Med Inc.) are newly developed diagnostic tests for rotavirus and norovirus infections. Methods Performance of AFIAS‐Rota/Noro assays was evaluated in comparison with RIDASCREEN® Rotavirus and Norovirus ELISA kits (R‐Biopharm) using clinical stool samples submitted from November 2018 to January 2019. Multiplex real‐time reverse transcription‐polymerase chain reaction was used as reference method. Results A total of 256 clinical specimens were analyzed. AFIAS‐Rota and RIDASCREEN Rotavirus had almost perfect agreement (Kappa value = 0.95), and substantial agreement was observed between AFIAS‐Noro and RIDASCREEN Norovirus (Kappa value = 0.80). For detection of rotavirus, AFIAS and RIDASCREEN assays showed satisfactory diagnostic sensitivity (100% and 97.8%, respectively) and specificity (99.5% and 99.1%). For detection of norovirus, the RIDASCREEN assay showed significantly higher sensitivity than the AFIAS‐Noro (86.0% and 66.0%, respectively; P = .002). Analytic specificity of AFIAS‐Rota/Noro assays showed no cross‐reactivity against any other bacteria (14 strains) or viruses (2 strains). Hands‐on time (6 minutes) and turnaround time (26 minutes) required to perform AFIAS assays were much shorter than those required for RIDASCREEN assays (20 and 150 minutes, respectively). Conclusion The AFIAS‐Rota/Noro assays showed overall excellent agreement with the RIDASCREEN assays. Although the AFIAS‐Noro assay exhibited lower sensitivity than the RIDASCREEN Norovirus assay for detection of norovirus, the AFIAS‐Rota/Noro assays could be useful as a rapid initial screening test in clinical laboratories due to its convenience and rapid turnaround time.


| INTRODUC TI ON
Acute gastroenteritis is one of the most impactful and common infectious diseases, accounting for millions of deaths annually in young children. Rotavirus and norovirus are leading causes of acute viral gastroenteritis spread through fecal to oral transmission. 1 Rotavirus infections are the primary cause of severe dehydrating gastroenteritis, especially in children below the age of five years. 2 Norovirus is the single most common cause of acute gastroenteritis in adults and the second major cause of severe diarrhea in infants and young children in the United States. 3 Patients with gastroenteritis are treated mainly with oral or intravenous rehydration solutions, and antibiotics are not routinely indicated in viral gastroenteritis. 1 Accurate test results are beneficial in managing patients, such as isolating patients to prevent transmission and prompting physicians to consider antibiotics therapy.
For appropriate treatment and infection control, accurate and timely identification of pathogens is necessary. Various diagnostic tools including electron microscopy, latex agglutination, immunochromatographic assay (ICA), enzyme immunoassays, and molecular assays have been developed. 4 Molecular methods such as reverse transcription-polymerase chain reaction (RT-PCR) are highly sensitive and specific. However, they are expensive and require specialized techniques and equipment. 4,5 On the other hand, ICA can be run individually, and ELISA assays can be easily performed without sophisticated equipment. 4,6 Thus, due to simplicity and swiftness, immunoassays including the ICA and enzyme-linked immunosorbent assay (ELISA) have been commercially used in routine clinical laboratories. 5 Here, we aimed to evaluate the performance of Automated

| MATERIAL S AND ME THODS
We used a total of 256 clinical stool samples submitted to the clinical microbiology laboratory at a tertiary referral hospital, from Inc.), and results were read after 12 minutes (min) using an AFIAS-6 scanner (Boditech Med Inc.). The scanner measured fluorescence intensity in the form of a relative cutoff index (COI) that was proportional to the concentration of the target antigens in the samples. The sample results were interpreted as "positive" when the COI of the AFIAS assays was ≥1.0, "negative" when COI was <0.9, or "indeterminate" when 0.9 ≤COI <1.0. 7,8 As a comparative method, rotavirus and norovirus antigen as-  Since the AFIAS-6 scanner has six channels, the HOT and TAT of the two assays were measured for six samples. HOT was defined as the time spent by a trained laboratory technician for preparing samples prior to equipment loading and detection. TAT was defined as the time interval between laboratory receipt of the sample and generation of the final result.

| RE SULT
Of 256 clinical stool samples, 46 were positive on both the AFIAS-Rota and RIDASCREEN-Rota assays ( Diagnostic specificity of AFIAS-Rota and RIDASCREEN-Rota was 97.6% (95% CI, 94.1%-99.1%) and 98.5% (95% CI, 95.5-99.6), respectively ( Table 2). Discordant results between the two assays were observed for 14 samples. A total of 11 results were positive by RIDASCREEN-Noro but negative by the AFIAS-Noro assay; 10 were confirmed to be positive by rRT-PCR. Although the two assays showed comparable specificity, the sensitivity of the RIDASCREEN-Noro assay was significantly higher than that of the AFIAS-Noro assay (P = .002).
The HOT (6 minutes) and TAT (26 minutes) required to perform the AFIAS assay were much shorter than those required for the RIDASCREEN assay (HOT, 20 minutes; TAT, 150 minutes). The time difference between the two assays originated primarily from cultivation time in the RIDASCREEN assay. Workflow of the major steps in the two assays is illustrated in Figure 1.

| D ISCUSS I ON
In this study, we evaluated AFIAS-Rota/Noro assays for detection of rotavirus and norovirus compared to the RIDASCREEN assays. For rotavirus, the AFIAS-Rota assay and RIDASCREEN-Rota assay were in good agreement and yielded satisfactory sensitivity (100% and 97.8%, respectively) and specificity (99.5% and 99.1%) compared to the rRT-PCR assay result. These results were comparable to other published studies for detection of rotavirus. 6 16,19 or the difference in analytical sensitivity among the assays. 20 Therefore, the AFIAS-Noro is recommended as an initial screening test. Patients clinically suspicious for norovirus infection despite initial AFIAS-negative results were indicated for reflex testing by molecular methods such as rRT-PCR. Nevertheless, AFIAS-Noro can be considered a useful initial screening test due to its simplicity and short TAT.
In conclusion, our data indicate that the AFIAS-Rota/Noro assays show overall excellent agreement with the RIDASCREEN-Rota/ Noro assays. Although the AFIAS-Noro assay exhibited lower sensitivity than the RIDASCREEN-Noro assay for detection of norovirus, the AFIAS-Rota/Noro assays could be useful as a rapid initial screening test in routine clinical laboratories due to their convenience and rapid TAT.

ACK N OWLED G M ENTS
This study was supported by Boditech Med Inc (Chuncheon, Korea).
The sponsor had no involvement in the study design, data interpretation, or writing of the manuscript.