Early Diagnosis of Tularemia by Flow Cytometry, Czech Republic, 2003-20151

Abstract

We retrospectively assessed the utility of a flow cytometry-based test quantifying the percentage of CD3+ T cells with the CD4-/CD8- phenotype for predicting tularemia diagnoses in 64 probable and confirmed tularemia patients treated during 2003-2015 and 342 controls with tularemia-like illnesses treated during 2012-2015 in the Czech Republic. The median percentage of CD3+/CD4-/CD8- T cells in peripheral blood was higher in tularemia patients (19%, 95% CI 17%-22%) than in controls (3%, 95% CI 2%-3%). When we used 8% as the cutoff, this test's sensitivity was 0.953 and specificity 0.895 for distinguishing cases from controls. The CD3+/CD4-/CD8- T cells increased a median of 7 days before tularemia serologic test results became positive. This test supports early presumptive diagnosis of tularemia for clinically suspected cases 7-14 days before diagnosis can be confirmed by serologic testing in regions with low prevalences of tularemia-like illnesses.

Keywords: Czech Republic; Francisella tularensis; ROC curve; T cells; bacteria; diagnosis; double-negative T cells; early diagnosis; flow cytometry; gamma delta T cells; intracellular pathogens; peripheral blood; tularemia; work-up; γδ T cells.

Figure 1

Flow cytometry gating strategy used to determine percentage of CD3+ lymphocytes that are CD3+/CD4–/CD8– T cells and γδ T cells in peripheral blood samples acquired from patients with suspected tularemia, Czech Republic, 2003–2015. A–C) Staining with CYTO-STAT tetraCHROME CD45-FITC/CD56-RD1 (phycoerythrin)/CD19-ECD/CD3-PC5, anti–CD4-Alexa Fluor 750, and anti–CD8-PC7 (Beckman Coulter, https://www.beckmancoulter.com). A) CD45 versus side scatter plot. Percentages of lymphocytes (red), monocytes (green), and granulocytes (blue) are indicated. In total, 3,000 lymphocytes were selected for further analysis. B) B cells (blue) and T cells (red) plotted according to their CD19 and CD3 expression. Percentages of cells within each quadrant are indicated. T cells were selected for further analysis. C) Percentage of CD3+ T cells not displaying CD4 and CD8 (CD4–/CD8–) determined with CD4 versus CD8 plots. Percentages of cells within each quadrant are indicated. D) Staining with anti–CD3-FITC and anti–T-cell receptor PAN γδ-PE (Beckman Coulter). After a side scatter and forward scatter plot (not shown), the percentage of lymphocytes that were CD3+/γδ T cells (green) were determined with a CD3 versus T-cell receptor pan–γδ plot. Percentages of cells within each quadrant are indicated. Flow cytometry was performed in the Immunology Laboratory of České Budějovice Hospital (České Budějovice, Czech Republic). ECD, phycoerythrin-Texas Red-X; FITC, fluorescein isothiocyanate; PC, phycoerythrin cyanine; PE, phycoerythrin.

Figure 2

Selection of tularemia cases (2003–2015) and controls (2012–2015) in investigation of whether γδ T cells or CD3+/CD4–/CD8– T cells can be used for early presumptive tularemia diagnosis, Czech Republic.

Figure 3

Correlation between percentage of CD3+ lymphocytes that are γδ T cells and percentage that are CD3+/CD4–/CD8– T cells in peripheral blood samples from patients with confirmed or probable tularemia diagnoses (n = 48), Czech Republic, 2003–2015. The Spearman correlation coefficient of this plot (0.830, 95% CI 0.679–0.906; p<0.0001) indicates a strong correlation and suggests that these T cells can be used interchangeably for tularemia diagnosis.

Figure 4

Percentage of CD3+ lymphocytes that are γδ T cells and CD3+/CD4–/CD8– T cells in peripheral blood samples from patients with confirmed or probable tularemia by clinical manifestation, Czech Republic, 2003–2015. The percentage of γδ T cells was determined for 48 cases and percentage of CD3+/CD4–/CD8– T cells for 64 cases. Paired comparisons (Kruskal-Wallis test) reveal no significant differences except for glandular versus typhoidal in γδ (p = 0.037) and CD3+/CD4–/CD8– T cells (p = 0.041). Boxes indicate interquartile ranges (IQRs), horizontal lines within boxes indicate medians, whiskers indicate range values <1.5× the IQR limits, and circles indicate outliers (i.e., values >1.5× the IQR limits).

Figure 5

Comparison of percentages of CD3+ lymphocytes with CD4–/CD8– phenotype in peripheral blood samples from patients with probable or confirmed tularemia cases (n = 64, 2003–2015) and controls (n = 342, 2012–2015), Czech Republic. Boxes indicate interquartile ranges (IQRs), horizontal lines within boxes indicate medians, whiskers indicate range values <1.5× the IQR limits, and circles indicate outliers (i.e., values >1.5× times the IQR limits). The percentage of CD3+/CD4–/CD8– T cells is significantly higher in cases than controls (Mann-Whitney U test, p<0.0001).

Figure 6

Receiver operating characteristic curve for diagnostic utility of raised CD3+/CD4–/CD8– T cells distinguishing probable and confirmed tularemia cases (n = 64, 2003–2015) from controls (n = 342, 2012–2015), Czech Republic. The area under the receiver operating characteristic curve is 0.970 (95% CI 0.952–0.988). The Youden index (circle on curve) is the maximal vertical distance (dashed line) of the curve from the diagonal line.

Figure 7

Comparison of time to first positive serologic test result for tularemia and time to raised CD3+/CD4–/CD8– T-cell percentage determined by flow cytometry relative to the time of symptom onset of 58 patients with probable or confirmed tularemia, Czech Republic, 2003–2015. Percentages of CD3+/CD4–/CD8– T cells >8% were considered raised. A positive serologic test result for tularemia was defined for probable cases as an antibody titer of >1:20 in any acute phase blood sample and for confirmed cases as a single antibody titer of >1:160 in any blood sample or a seroconversion from negative to positive (any titer) or a 4-fold increase in titer between acute and convalescent patient samples (agglutination test; Tularemia Diagnostic Set, Bioveta a.s., https://www.bioveta.eu). Boxes indicate interquartile ranges (IQRs), horizontal lines within boxes indicate medians, whiskers indicate range values <1.5× the IQR limits, and circles indicate outliers (i.e., values >1.5× times the IQR limits). The CD3+/CD4–/CD8– T cells increased before Francisella tularensis–specific antibody titers increased (Wilcoxon signed rank test, p<0.0001).