Abstract

1. C. M. Farris1, B. Kiehl2, J. Samuel3 1Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, MD, 2GenBio, San Diego, CA, 3Department of Microbial and Molecular Pathogenesis, College of Medicine, Texas A&M, Bryan, TX Comparison between indirect immunofluorescence antibody (IFA) and dotblot enzyme immunoassay (EIA) methods to detect Coxiella burnetii (Q Fever) phase I and II antibodies Conclusions Abstract Methods & Materials Sensitivity • No significant difference between IFA and the ImmunoDOT dotblot method is detected. • Unlike the IFA method, ImmunoDOT uses cell-free Coxiella burnetii Phase I and II antigens. • The improved purity of the Phase I antigen may allow earlier detection. • Although the Phase II antigen purity is improved, clinical interpretation using Phase II results is not affected because Q fever is endemic in most populations. That is, approximately one-third of the population expresses a low level of Coxiella burnetii Phase II antibody. • ImmunoDOT offers the advantage that only one test is required and the test method can be performed in moderate complexity laboratories. Coxiella burnetii is the causative agent of Q Fever, a world-wide zoonotic disease. Diagnosis of human disease is typically made using serology. Comparison between the indirect immunofluorescence antibody (IFA) and dot blot enzyme immunoassay (EIA) methods is reported. Materials and Methods: A commercial dotblot immunoassay, ImmunoDOT™ Coxiella Burnetii, was developed using a cell-free culture method to prepare purified phase I and II Coxiella burnetii antigens. IgG and IgM commercial IFA kits (Focus Diagnostics™) and in-house prepared IFA tests are compared to the dotblot kit. Paired sera collected from thirty-eight (38) patients with symptomatology and laboratory results consistent with C. burnetii are tested. These serum pairs are part of a C. burnetii reference serum panel maintained by the university laboratory. Each of the 76 specimens (38 serum pairs) is tested using the IFA tests detecting Phase I and II IgG or IgM. The 76 specimens are also tested using the ImmunoDOT Coxiella Burnetii test. Results: Sensitivity, based on paired serum interpretation is shown in Table 1. There is no significant difference (p=0.05, Chi-square, confidence limits based on Gart and Nam's score method with skewness correction) between assay methods. Table 1: Sensitivity Summary: No significant difference between IFA and the dotblot is detected. In several cases, a second convalescent specimen is required for IFA sero-diagnosis while EIA reports a positive result using only a single, acute specimen. Presumably, phase I antigen purity improvement contributes to the added diagnostic utility. To evaluate sensitivity, each of the 76 specimens (38 serum pairs) were tested using three serology tests. All testing was performed at the university laboratory. ImmunoDOT interpretation was made following package insert instructions. IFA interpretation was made using the following criteria. There is no significant difference between assay methods (p=0.05, Chi-square, confidence limits based on Gart and Nam's score method with skewness correction). Specificity Using the ImmunoDOT dotblot method, forty-seven presumptive negative specimens collected from healthy, U.S. subjects are tested. Using package insert instructions, specificity is 100% (47/47). Sixteen (34%) of these clinical negative specimens reported borderline reactivity (see below). Reference Omsland, et al, 2009, Host cell-free growth of Q fever bacterium Coxiella burnetii, Proc Natl Acad Sci USA, 106 (11):4430-4434. Paired Serum Specimens: Serum pairs collected from thirty-eight (38) patients with clinically defined Q Fever were tested. The serum pairs are part of a Q fever reference serum panel maintained by the university laboratory. IFA Method: Two indirect immunofluorescence assay (IFA) methods were evaluated. One is a commercial test (Focus Diagnostics, detecting IgG and IgM Phase 1 and Phase 2 antibody types) and the other was produced by the university laboratory using the same antigen. The serial dilution schemes of the assays differ slightly. For the university method, antigen is fixed to the slide using acetone. The procedures are the same for both methods following the commercial test’s package insert instructions. Dot Blot EIA Method: A commercial dotblot immunoassay (ImmunoDOT™ Coxiella Burnetii) was developed using Phase I or Phase II antigens prepared from Coxiella burnetii propagated using a cell-free culture method (1). Since the dotblot format provides a multiplex result, only one test is required. Presumptive Negative Specimens: To evaluate ImmunoDOT specificity, forty-seven (47) sera collected from prospective U.S. blood donors were tested. Introduction Q fever, caused by Coxiella burnetii, is a world-wide zoonotic disease. Infection usually occurs by aerosol transmission in a rural (farm, back country, etc.) environment. Like many respiratory infections, many are asymptomatic. Disease usually presents as flu-like symptoms that may progress to atypical pneumonia or acute respiratory distress syndrome (ARDS). Fever may last 1-2 weeks. The time from infection to disease expression is several days coinciding with antibody production. Sero-diagnosis is the most common laboratory method. Historically, the indirect immunofluorescence antibody (IFA) method has been the most commonly used test and remains the gold standard for diagnosis. Comparison to a dot blot enzyme immunoassay (EIA) method (ImmunoDOT™) is reported. Disclaimer The views expressed in this article are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. Contact Information Bryan Kiehl (bkiehl@GenBio.com) Mobile (+1 760-855-8999)