Materials and Methods
These researchers utilized eighty-two serum samples and thirteen cerebrospinal fluid samples. An IFA technique, involving a fluorescein-labeled conjugate, was used to visualize these samples. They were examined by a transmitting fluorescent microscope with a one-hundred watt mercury lamp. Fluorescence was then analyzed visually with a qualitative scale, which measured the cytoplasmic fluorescence. Both positive and negative controls were utilized for each assay, and each was performed in triplicate. For a specific protocol please refer to the original paper (Malan et al. 2003).
In order to compare the IFA results to those obtained by ELISA protocols, three external laboratories were consulted for analysis. These laboratories developed the reference ranges used in the study. Cross reactivity to other related viruses was also analyzed through comparison to eighteen positive controls for Saint Louis Encephalitis (SLE), WNV, and dengue. Statistical analysis of the results was performed with the use of two by two contingency tables.

Results
There was a ninety-eight percent agreement between the ELISA analysis and the IFA analysis. Sensitivity was documented to be ninety-six percent, and specificity was shown to be 100%. IFA was shown to be only slightly less sensitive than MAC-ELISA testing. The IFA was not sensitive enough to determine the difference between antibodies to SLE, WNV, and dengue. The authors, however, note that the test can be used along with other personal information obtained from the patient in order to determine the particular virus involved in the patient’s symptoms.

Discussion
Detection of IgM and IgG with the IFA protocol developed by this paper was shown to be cost effective and nearly as sensitive as MAC-ELISA. The difficulties of performing the MAC-ELISA protocols were overcome with this IFA method. The IFA protocol can be performed in laboratories that do not have authorization to store actual WNV. This assay is also well suited for laboratories that have limited operational budgets.