Evaluation of two current generation enzyme immunoassays and an improved isolation-based assay for the rapid detection and isolation of rotavirus from stool.
BACKGROUND
Rapid and accurate rotavirus testing is important in decisions involving patient care and management. Quality assurance testing needs to be periodically performed, especially among widely used assays having a direct impact on patient care.


OBJECTIVE
To evaluate the current generation Kallestad Pathfinder Direct antigen Detection system (PTH), and the widely used Rotaclone(R) Rotavirus EIA Diagnostic Kit (RTC), in comparison with an improved cell culture amplification-antigen detection (CCA-Ag) isolation-based assay.


METHODS
Two hundred stool specimens (specimen stored at > or =-75 degrees C), which had been previously tested by PTH, were tested by RTC and CCA-Ag. Discordant specimens were retested by PTH, blocking assay, polyacrylamide gel electrophoresis (PAGE), and/or electron microscopy (EM).


RESULTS
Among 200 stool specimens, 197 were in accord by PTH, RTC and CCA-Ag. The sensitivity, specificity, positive and negative predictive values for RTC, PTH and CCA-Ag were, 100, 99, 99, 100, 100, 99, 99, 100; and 98, 100, 100, 98%, respectively. Among five initially discordant specimens, two required a period of 10 days to affect isolation. A non-cultivatable (CCA-Ag negative) but true positive specimen, was identified as rotavirus group A serotype G2 by RT-PCR. Four true positive but discordant specimens were blocking assay negative using one or both EIA kits.


CONCLUSIONS
PTH and RTC are excellent rotavirus detection system. However, PTH is more expensive (ca. $3.50 vs. $2.00 per test), mandates a slightly longer turn-around time (ca. 1 vs. 1.5 h), and necessitates slightly more hands-on manipulative/preparative steps. Blocking assay was not a reliable confirmatory test for the resolution of specimen discordancy. A combination of CCA-Ag, PAGE, EM, and/or perhaps RT-PCR, is recommended as an appropriate test panel for the resolution of discordant results during assay evaluation. The newly modified and simplified 48-h rotavirus isolation-based assay may serve as a base line methodology in laboratory evalaution studies, as a laboratory support methodology during drug/vaccine efficacy trials, or for the testing of sources (e.g., biopsy/autopsy tissues) not approved for assay by commercial rotavirus kits.
Departments of Laboratories, North Shore University Hospital - NYU School of Medicine, Manhasset, NY, USA. montmor@aol.com
S M
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Lipson
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Svenssen
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L
Goodwin
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Porti
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Danzi
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Pergolizzi
D000956:Antigens, Viral / Q000302:isolation & purification; D003365:Costs and Cost Analysis; D005243:Feces / Q000821:virology*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000191:economics / Q000379:methods*; D011933:Reagent Kits, Diagnostic / Q000191:economics; D015203:Reproducibility of Results; D012401:Rotavirus / Q000276:immunology / Q000302:isolation & purification*; D012400:Rotavirus Infections / Q000821:virology*; D012680:Sensitivity and Specificity
D000956:Antigens, Viral; D011933:Reagent Kits, Diagnostic