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This evaluation was performed on all human specimens submitted for WNV testing for the province, including West Nile fever WNF cases, using an expanded case definition incorporating molecular diagnosis to provide data on the fullest range of clinical presentations. We thank Alberta Health and Wellness for providing financial support for WNV testing and for clinical case definitions. Specificity was high for the Focus kit at West Nile virus WNV has spread rapidly across North America since its introduction in , creating a need for rapid and accurate laboratory diagnosis 3 , 8. This delayed antibody response is more common than in most earlier reports 9 , 10 , This difference may be due to the comprehensive population-based format of this study, which included a high percentage of patients with WNF. Phone: Fax: E-mail: ac. Jayaraman , 3 Kevin Fonseca , 1 Michael A. For IgG-positive patients, the IgG test was repeated on the acute- and convalescent-phase specimens in the same run. Discrepant results between the two kits were reported as indeterminate. Abstract West Nile virus WNV has spread rapidly across North America, creating a need for rapid and accurate laboratory diagnosis on a large scale. Gayatri C. The remaining 22 serum pairs all showed a static HI titer of or greater, median, 1, Ultimately, 2, sera were received from a total of 2, Alberta residents, of whom were considered to have confirmed WNV infections and were probable cases. TABLE 1. This article has been cited by other articles in PMC. Consolidated human WNV diagnostic testing for the province of Alberta, Canada, at the public health laboratory permitted a large-scale evaluation of the assays, covering a wide clinical spectrum. In this study, the first two available WNV IgM EIA kits were employed prospectively on a population basis and evaluated in light of both previously available HI titers and new molecular assays. A laboratory testing information bulletin was provided to all physicians and was posted on the laboratory website. The use of nucleic acid testing identified many cases on the first blood collection and partially compensated for the lack of follow-up sera. When specimens were received without history, the blank form was faxed to the physician for completion. Tilley et al. A clinical history form was circulated and placed on the website to assist with specimen triage and communication and to provide travel and vaccination history for interpretation of results. As these tests could not be performed on all patients, a probable case definition was also used. West Nile virus diagnostic tests were conducted at one site of the Provincial Laboratory for Public Health. The test algorithm is shown in Fig. The performance of IgM assays in upcoming years remains to be seen, given the recent report of persistent IgM positivity in many patients using in-house tests It is likely that changing titers will need to be demonstrated to confirm an acute diagnosis in future years. Journal List J Clin Microbiol v. National Center for Biotechnology Information , U. Consolidation at one site provided standardized testing of sufficient volume to evaluate test performance and provided comprehensive reporting to public health epidemiologists. A preliminary validation was performed for the first cases for which IgM was positive in both commercial assays. As convalescent-phase sera were submitted in a minority of cases, a limitation of this study is that some cases with delayed seroconversion may have been missed. A twofold increase in IgG index proved to be due to acute flavivirus infection in all cases. Immunoglobulin M IgM capture enzyme immunoassays EIA became commercially available in the summer of , but limited data are available on their clinical performance. It remains possible, however, that the sensitivities of the IgM kits may be lower than reported here if there were undetected cases. In some patients with WNV infection and previous exposure to flaviviruses, the IgM response is suppressed and only an IgG response is seen 3. Specificity is high for the Focus kit, and for the Panbio kit when background subtraction is employed. IgG test result No.{/INSERTKEYS}{/PARAGRAPH} Drebot , 4 Jutta Preiksaitis , 2 and Julie Fox 1. TABLE 2. Specimens recommended for testing were acute- and convalescent-phase sera, whole blood EDTA , and cerebrospinal fluid CSF , where appropriate. The specificity in the presence of other acute flavivirus infections cannot be determined from our population. The sensitivity of the IgM assays is high, especially for neurological cases, provided that follow-up sera are collected after the first week of illness. IgM assays were repeated on all convalescent-phase sera using both kits. Michael A. A subsequent version of the kit, with a subtraction control well for each specimen, corrected this problem, without compromising the test sensitivity. At the end of the season a second-generation WNV IgM kit became available Panbio-2 , which incorporated a control well for each serum specimen. Hemagglutination inhibition titers were performed against West Nile and St. Historically, hemagglutination inhibition HI assays have been used for the serological diagnosis of arboviral infections 1. The IgM index was calculated for each specimen by dividing the patient serum optical density reading by a standard cutoff reagent reading, performed with every run, as per the manufacturers' instructions. Indices were calculated by division of patient serum optical density by kit control optical density, as per the manufacturers' instructions. Peter A. Eight patients had insufficient testing for categorization. In-house IgM tests have been described previously for diagnosis of arboviral infections in the United States and Europe 11 , Commercial kits for WNV IgM have only recently become available and offer the possibility of rapid and larger-scale testing but have not been well evaluated in the literature 5. All convalescent-phase IgM-negative sera were screened for IgG, and 10 were positive. In Alberta, WNV serological and molecular testing was implemented at a single public health laboratory site to serve the province in The availability of the commercial assays during the first season of WNV activity provided an opportunity for the first large-scale, head-to-head evaluation of the kits in a previously naive population, where other flaviviruses are nonendemic. Reagents for the IgM assay IgM antibody capture enzyme-linked immunosorbent assay were made available for a period to public health laboratories within the United States 6 but were not widely available in other countries. Patients were considered probable WNV cases if i the WNV IgM test was positive by both commercial kits and was subsequently positive by the second-generation Panbio kit incorporating a background subtraction step or ii the WNV HI test showed a fourfold rise in titer in the absence of recent travel. The control well did not contain specific antigen, and the optical density for this well was subtracted from the WNV antigen well to control for nonspecific binding. Two-week follow-up sera were requested for all patients. These assays have therefore proven clinically useful for timely West Nile diagnosis in a region where St. This bulletin recommended testing patients with neurological presentations, patients with fever who had blood or organ donation or receipt, and immunocompromised patients with fever. West Nile virus was first reported in Alberta on 9 July , having been found in a dead magpie, and the first human case was detected 9 August by PCR of a blood donor specimen. Acute- and convalescent-phase sera were run concurrently. Anamnestic responses therefore appear to be a rare problem for the IgM tests in our population. J Clin Microbiol. In summary, new commercially available enzyme immunoassays proved to be useful clinical tools in the inaugural WNV season in the province of Alberta, a region nonendemic for other flaviviruses. Test result No. Thus, if serology is used as the sole method for WNV testing, it is important to stress to ordering physicians that a follow-up specimen, collected after the first week of illness, is required for reliable serological diagnosis. Two thousand nine hundred sixty-nine sera were tested, from 2, Alberta residents, and cases were identified. Plaque reduction neutralization titers PRNT are specific, but require containment level 3 viral culture and are laborious to perform. These are not the same group of noncases tested by EIA above. Acute- and convalescent-phase IgG index values, calculated relative to the kit cutoff control as per the manufacturers' instructions, were compared to HI titers to determine whether IgG could be used quantitatively and serve as a replacement for HI. Louis encephalitis, Japanese encephalitis, and dengue are nonendemic. One acute dengue case had a rising index but remained just below the IgG-positive cutoff point. Screening of asymptomatic blood or organ donors is not included in the study. Of the seven patients who did not show an IgG response, three had convalescent-phase sera collected less than 14 days after the onset of illness. This technique is broadly cross-reactive among the flaviviruses and requires confirmatory testing for specific viral diagnosis. A sensitive method utilizing nucleic acid sequence-based amplification NASBA was used as a first-line assay for plasma samples. Few reports have been published on their performance, and these provide limited data on comparison with the in-house Centers for Disease Control and Prevention assay 5. Louis encephalitis viruses on acute- and convalescent-phase serum pairs using antigens prepared from suckling mice 1. {PARAGRAPH}{INSERTKEYS}West Nile virus WNV has spread rapidly across North America, creating a need for rapid and accurate laboratory diagnosis on a large scale. Open in a separate window. Two different nucleic acid amplification methods were employed for detection of WNV-specific RNA in extracted nucleic acid, each targeting a different portion of the WNV genome. The IgG index was calculated by division of the patient serum optical density value by that of the cutoff reagent standard performed with each run, as per the manufacturer's instructions. The IgG index data are summarized in Fig. Author information Article notes Copyright and License information Disclaimer. Of these, no additional WNV cases were identified by rising HI titers; all had stable low flavivirus titers, and the results were considered to reflect past flavivirus infections. Mean ages of cases and noncases were Gender was not significantly different between cases and noncases Sensitivity of the Focus kit was The Panbio-1 kit had a similar sensitivity, at The kinetics of the Panbio-1 IgM kit are nearly identical data not shown. As seen in the HI assay, stable high IgG indices did not differentiate between recent and past flavivirus infections.