The accurate diagnosis of new dengue virus infections requires both virus detection and detection of IgM anti-dengue because of the 4 - 5 day window after onset of fever when the IgM test is negative. While diagnostic tests for IgM anti-dengue are commercially available, virus detection by cell culture, nucleic acid detection (e.g., PCR, NASBA [Nucleic Acid Sequence Based Amplification]) or detection of NS1 antigen in serum require the capacity of a research laboratory that produces the required reagents and performs the tests under good laboratory practices.

The plaque reduction neutralization test (PRNT) is the only assay available to measure immunity to dengue virus infection and reliably differentiate primary from secondary infections. However, the Kingdom, PDVI attempted to produce an International Reference Standard that would allow PRNT results to be expressed in international units (IUs) and improve comparison of results between laboratories worldwide. However, a multi-laboratory, comparison of the proposed international reference reagents showed an unacceptably high level of test variation. PRNT is performed only in research and reference laboratories, has not been standardized between laboratories, and is not easily adapted to handle large numbers of specimens. To better standardize performance of the PRNT, the PDVI held a training workshop in July 2004 for senior technicians from Asia and the Americas at Mahidol University Center for Vaccine Development. Standardized protocols, methods and reagents were transferred to the participants through hands-on performance of the PRNT. In collaboration with WHO and the National Institute for Biologic Standards and Control (NIBSC), United

There appears to be increasing interest in commercial production of dengue diagnostic tests. Working with TDR, a series of consultants meetings was held, which included diagnostics manufacturers, to identify needs in this area. Of high priority is the availability of a well characterized evaluation panel to determine the performance of current or future diagnostic tests as well as the performance of laboratories doing these tests. To develop this evaluation panel, TDR has designated and funded two Dengue Reference Diagnostics Laboratories and seven Diagnostics Evaluation Laboratories. A TDR-PDVI consultative workshop was held in July 2005 and developed and implemented a plan to produce a Dengue Evaluation Panel to evaluate diagnostic tests for acute dengue virus infection, which should become available in the last quarter of 2006.

An important consideration in the evaluation of dengue vaccines is that the PRNT, although the only test available, may not provide the best measurement of protective immunity to dengue virus infection. For instance, human challenge studies have shown that some persons are susceptible to infection and disease with the same dengue serotype for which they are PRNT-antibody positive, and some persons negative for PRNT type-specific antibody do not develop disease when challenged with that dengue virus serotype. PDVI is funding pre-clinical research to characterize the types and binding sites of antibodies involved in virus neutralization to develop a better test for dengue immunity. This work has been greatly facilitated by ongoing work on West Nile Virus, another Flavivirus, to answer the same questions. It is anticipated that the results of this research will lead to development of improved diagnostic tests for immunity (natural or vaccine induced) to dengue. The PDVI is prepared to ensure the further development and evaluation of these prototype tests.

A concern during evaluation of dengue vaccines is that immunization could immunologically prime a vaccine recipient and cause enhanced dengue disease upon later exposure to wild-type virus infection. While there may be some scientific disagreement as to the frequency with which immune enhanced disease occurs, its presence seems well established. Most studies point to antibodies as the primary mechanism of immune enhancement or ADE. What is missing is information about the type or level of antibody(ies) involved and an assay to measure these antibodies. Having an assay to detect ADE-related antibodies would provide an important tool to differentiate vaccine associated ADE from naturally occurring disease. In addition, if ADE is observed following immunization, knowledge about the characteristics of the antibodies involved could accelerate modifying vaccines to eliminate this problem.