Morphological and cytokine profiles as key parameters to distinguish between Gram-negative and Gram-positive bacterial keratitis

Abstract

Bacterial keratitis (BK) is an ocular disorder associated with poor visual prognosis. Quantification of the associated inflammatory response may provide insight into the pathogenesis of BK and guide treatment options. In this exploratory study, we evaluated 45 BK patients and 20 healthy controls by optical coherence tomography and pro-inflammatory tear cytokine analysis. The aim was to quantify the differential morphological and cytokine inflammatory response between Gram-negative and Gram-positive BK and to determine the diagnostic value of corneal thickness (CT) and infiltrate thickness (IT) in distinguishing Gram-ve BK in a clinical cohort. Greater CT and IT, at clinical presentation, were indicative of Gram-ve infection with values detected of ≥ 950 μm and ≥ 450 μm, respectively. Combination of these CT and IT values had a 100% sensitivity and 83.3% specificity as a diagnostic indicator of Gram-ve infection. Similarly, there were higher levels of IL-1β, IL-6 and IL-8 cytokines were quantified in keratitis caused by Gram-negative bacteria. Among the different tear cytokines analysed, a significant reduction after three days of treatment was detected for pro-inflammatory cytokines IL-1β, IL-2, IL-6, IL-8 and TNF-α, prior to starting with the administration of steroid drops. Overall, this study shows the potential value of serial OCT and tear cytokine measurements in the management of BK.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Anterior segment optical coherence imaging protocol for bacterial keratitis (a–c) and controls (d, e). (a) Picture of bacterial keratitis, illustrating the anterior segment optical coherence tomography (AS-OCT) imaging protocol with the same axis high resolution scan carried out at presentation and after resolution of infection. (b) An AS-OCT scan at presentation, illustrating the measurement of corneal thickness (810 μm) and infiltrate thickness (270 μm) at presentation. (c) Measurement of final corneal thickness (440 μm) once the infection has resolved. (d) Four-quadrant AS-OCT scans of the control healthy cornea. The flap tool was used to identify a central 4 mm area, a mid-peripheral area defined by the 4 mm zone and an outer 8 mm zone, and a peripheral area extending from the 8 mm zone to the limbus. The corneal thickness was measured in the centre of each area on all four scans with calliper tools and the mean corneal thickness of all patients plotted. (e) The plotted corneal thickness maps of healthy control subjects.

Figure 2

Comparison of AS-OCT quantification parameters between Gram−ve, Gram+ve and microbiology negative bacterial keratitis. (a) There was a significant difference in presentation corneal thickness between Gram−ve and Gram+ve groups, and between Gram−ve and microbiology negative groups, but not between Gram+ve and microbiology negative groups. (b) There was a significant difference in presentation infiltrate thickness between Gram−ve and Gram+ve BK, and Gram−ve and microbiology negative BK, but not between Gram+ve and microbiology negative BK (p = 1.0). (c) There was a significant difference in corneal tissue swelling between Gram−ve and Gram+ve groups, and Gram−ve and microbiology negative groups, but not between Gram+ve and microbiology negative groups. (d) There was a borderline significant difference in corneal tissue loss between Gram−ve and Gram+ve BK (p = 0.12), a significant difference between Gram−ve and microbiology negative BK, and no significant difference between Gram+ve and microbiology negative BK.

Figure 3

Quantification of the cytokine and chemokine levels present in tears. Comparison of cytokine and chemokines levels (pg/ml) present in tears between bacterial keratitis (BK) and control (a) and Gram−ve versus Gram+ve bacterial keratitis (b) at presentation are shown in the plots. All cytokines/chemokines were elevated at presentation of bacterial keratitis compared to controls, except for IL-12p70. The levels of IL-1β, IL-6 and IL-8 were significantly greater in Gram−ve than Gram+ve bacterial keratitis. Asterisks over the bars denote statistical significance (p < 0.05).

Figure 4

Time course cytokine and chemokine change in resolving bacterial keratitis. Quantification of IL-1β, IFN-γ, GM-CSF, IL-10, IL-12, IL-2, IL-6, IL-8 and TNF-α levels (pg/ml) at presentation and after 3, 4 and 14 days of treatment. A statistically significant reduction was found for IL-1β (p = 0.034), IFN-γ (p = 0.037), IL-2 (p = 0.04), IL-6 (p < 0.001), IL-8 (p = 0.004) and TNF-α (p = 0.008); a reduction of borderline statistical significance was found for GM-CSF (p = 0.068), IL-10 (p = 0.059) and IL12p70 (p = 0.068). The mean [SD] and median [IQR] values of each cytokine concentration, as well as the levels of their induction in BK compared to control tears (BK/control) at presentation and after 3, 7 and 14 days of treatment are specified in the Supplementary Table 3.