Comparison of enzyme-linked immunosorbent assay, Western blotting, microagglutination, indirect immunofluorescence assay, and flow cytometry for serological diagnosis of tularemia

Abstract

The serodiagnostic efficiencies of five different approaches to detecting antibodies (immunoglobulins G, A, and M) developed in clinically proven infections with Francisella tularensis have been assessed. Fifty serum samples from patients suffering from tularemia during an outbreak in Sweden were compared with samples from 50 healthy blood donors (controls) by using an enzyme-linked immunosorbent assay (ELISA), microagglutination (MA), Western blotting (WB), an indirect immunofluorescence assay (IIFA), and flow cytometry (FC). ELISA, WB, and FC were based on the use of preparations of lipopolysaccharides (LPS) of the live vaccine strain of Francisella tularensis subsp. holarctica (ATCC 29684) as a capture antigen. Whole methanol-fixed bacteria were used for IIFA and MA. Optimized protocols yielded a diagnostic sensitivity and specificity of 100% for WB, MA, and FC, 98% for ELISA, and 93% for IIFA. A total of 6,632 serum samples from individuals between the ages of 18 and 79 years, representatively recruited from all regions of Germany, were screened to estimate and confirm the positive predictive value (PVpos) of the ELISA. Serum samples from 15 (0.226%) individuals tested positive for F. tularensis-specific antibodies by ELISA and confirmatory WB. The resulting prevalence-dependent PVpos of 10.2% and specificity of 98.1% were consistent with our findings for tularemia patients and controls. We conclude that the combined usage of a screening ELISA and a confirmatory WB based on LPS as a common antigen, as well as the MA, is a suitable serodiagnostic tool, while the quality of the IIFA is hampered by subjective variations of the results. FC is a promising new approach that might be improved further in terms of multiplex analyses or high-throughput applications.

FIG. 1.

Distribution of F. tularensis LPS-specific immunoglobulin levels. Levels in sera of patients (n = 50) and healthy controls (n = 50) were determined by the methods given above the plots. Sera for ELISA were diluted 1/640.

FIG. 2.

Determination of F. tularensis antibodies by flow cytometry. Representative histograms of green fluorescence intensities of five serum samples from tularemia patients and two from healthy controls are shown.

FIG. 3.

Comparison of ELISA and flow cytometry methods. A scatter plot of mean fluorescence intensities obtained by flow cytometry and optical densities obtained by ELISA is shown. Linear regression analysis assessing the association of test results obtained for patients' sera (n = 50) by flow cytometry with those obtained by ELISA is indicated. Dashed lines, 95% confidence intervals of the regression function. Sera for ELISA were diluted 1/640.

FIG. 4.

Tularemia prevalence-dependent predictive values of ELISA. Positive (filled circles) and negative (open circles) predictive values of ELISA for a hypothetical range of disease prevalence were calculated based on the results given in Table 1. Ninety-five percent confidence intervals are indicated.

FIG. 5.

Survey of serum anti-F. tularensis antibody levels in Germany (n = 6,632). The distribution of ELISA results stratified by a cutoff level of 0.200 (line a) and results of confirmatory Western blotting are shown. Line b indicates the threshold value of confirmed positive samples. For calculation of specificity, sera with values between lines a and b (n = 18) were considered true negatives by ELISA, since all of them were negative by Western blotting. All serum samples were diluted 1/250. The cutoff was determined by 200 independent measurements of the negative-control serum and calculation of the range of values equal to 3 times the standard deviation. For a better overview, results for only 1/50 of the serum samples without subsequent Western blotting, randomly selected, are given.