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Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus
Published on: January 28, 2019
Utility of Cell Culture-Based Immunocolorimetric Assay for Detection of Rotaviruses in Fecal Specimens Collected From
Neelakshi S Kumbhar1, Supriya S Suryawanshi1, Prabha Hela1
1ICMR-National Institute of Virology, Pune, Maharashtra, India.
Insights
A new cell culture-based rotavirus immunocolorimetric assay (RV-ICA) detects live rotavirus. This method aids in identifying samples for rotavirus isolation, offering an advantage over existing diagnostic techniques.
Area of Science:
- Virology
- Infectious Diseases
- Diagnostic Assays
Background:
- Rotavirus (RV) is a primary cause of severe gastroenteritis in children globally.
- Accurate and timely diagnosis of rotavirus infection is crucial for effective treatment.
- Current diagnostic methods, real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA), have limitations.
Purpose of the Study:
- To develop and validate a novel cell culture-based rotavirus immunocolorimetric assay (RV-ICA).
- To evaluate the sensitivity and specificity of the developed RV-ICA compared to established methods.
- To assess the utility of RV-ICA for the isolation of live rotavirus from clinical specimens.
Main Methods:
- A rotavirus immunocolorimetric assay (RV-ICA) was developed using the MA104 cell line, known for supporting rotavirus growth.
- The RV-ICA was standardized and validated using 84 fecal specimens from children with acute gastroenteritis.
- Results from RV-ICA were compared against commercial Ag-ELISA and in-house RT-qPCR.
Main Results:
- The cell culture-based RV-ICA was successfully standardized and validated.
- RV-ICA demonstrated a sensitivity of 90.7% and specificity of 36.6% when compared to commercial Ag-ELISA.
- Compared to RT-qPCR, RV-ICA showed a sensitivity of 84.3% and a specificity of 57.1%.
Conclusions:
- The developed cell culture-based RV-ICA effectively detects live rotaviruses in fecal specimens.
- This assay offers an advantage over Ag-ELISA and RT-qPCR by enabling the isolation of rotavirus.
- RV-ICA facilitates the identification of samples suitable for rotavirus isolation, aiding further research and diagnostics.
Background:
Rotavirus (RV) is the leading cause of severe gastroenteritis in children and is responsible for significant morbidity and mortality in developing and low-income countries. Hence, precise and prompt diagnosis of rotavirus infection is vital for treatment. The currently employed detection methods include rotavirus real-time reverse transcriptase polymerase chain reaction (RT-qPCR) and antigen-capture enzyme-linked immunosorbent assay (Ag-ELISA) which have some known strengths and weaknesses.
Methods:
Rotaviruses are difficult to grow in various cell substrates; however, a few cell lines, including MA104, show evident virus growth. Hence, MA104 cell-based immunocolorimetric assay (ICA) was developed, and its sensitivity and specificity were studied with the existing rotavirus detection methods that target the viral antigen or nucleic acid. A total of 84 fecal specimens obtained from acute gastroenteritis children were utilized, and the results of ICA were compared with commercial Ag-ELISA and in-house RT-qPCR.
Results:
A cell culture-based RV-ICA was successfully standardized and validated with commercial Ag-ELISA and in-house RT-qPCR. The sensitivity and specificity of ICA with commercial Ag-ELISA were found to be 90.7% and 36.6%, respectively. The comparison of RT-qPCR with RV-ICA showed slightly lower sensitivity, that is, 84.3%, whereas increased specificity, that is, 57.1%. The positive predictive value of RV-ICA with Ag-ELISA and RT-qPCR was 60% and 90.7%, respectively. It is well accepted that RT-qPCR is a more sensitive and rapid assay for virus detection due to amplification and quantification of target nucleic acid. However, a cell culture-based ICA has an added advantage over Ag-ELISA and RT-qPCR, due to added possibility of rotavirus isolation from positive fecal specimens.
Conclusions:
The standardized cell culture-based RV-ICA supports the detection of live rotaviruses from fecal specimens over the commercial Ag-ELISA and RT-qPCR, thus helps identify samples suitable for rotavirus isolation.
