A need for standardized rabies-virus diagnostic procedures: Effect of cover-glass mountant on the reliability of antigen detection by the fluorescent antibody test

The direct fluorescent antibody test is a sensitive and specific procedure used in the routine diagnosis of rabies. However, given the critical role of the rabies diagnostic laboratory in patient management and public health decision-making, the use of a standardized national rabies diagnostic procedure is highly recommended. Seemingly small variations in test procedures may have dramatic effects on sensitivity. For example, two independent reports of diminished staining performance of two lots of a commercial anti-rabies conjugate were investigated in this study. The diminished staining occurred only with a single rabies-virus variant, associated with big brown bats, Eptesicus fuscus, in the southwestern United States. Similarly diluted and prepared diagnostic reagents provided bright staining on all other variants of rabies-virus tested. Subsequent evaluation disclosed that the phenomenon was associated with the relative concentrations of glycerol used in the mounting media by the reporting laboratories. These findings, related to the proper selection of an optimal cover-glass mountant for use in the immunofluorescence procedure, demonstrate the potential for erroneous results with severe implications for patient health, when uncontrolled variations in protocol occur. This paper underscores the necessity for all rabies diagnostic laboratories to follow one standard protocol. Such a protocol has been placed on the websites maintained by the Centers for Disease Control and Prevention: http://www.cdc.gov/ncidod/dvrd/rabies/professional/publications/DFA_diagnosis/DFA_protocol-b.htm.