The Diagnostic Value of Triggering Receptor Expressed on Myeloid Cells-1 in Post-Traumatic Bacterial Endophthalmitis

Abstract

Purpose: To determine whether soluble-triggering receptor expressed on myeloid cells-1 (sTREM-1) could serve as a reliable diagnostic biomarker of post-traumatic bacterial endophthalmitis (PTBE).

Methods: Thirty-two patients (32 eyes) clinically diagnosed having PTBE were further divided into a culture-positive (CP) group and a culture-negative (CN) group. Sixty-two patients (62 eyes) without traumatic endophthalmic infection were also enrolled. Twenty-one eyes from 11 donors without globe ocular injuries were included as control group. Vitreous sTREM-1 levels were detected by ELISA. The expression and tissue distribution of TREM-1 were revealed by immunohistochemistry. The diagnostic value of sTREM-1 was determined by receiver operating characteristic curve (ROC). The correlation between sTREM-1 concentration and final best-corrected visual acuity (FBCVA) and Peyman endophthalmitis score (PES) were also assessed.

Results: The vitreous sTREM-1 level in the PTBE group was higher than that in noninfected group and control group (P < 0.05). No remarkable difference was found between the CP group and the CN group in vitreous sTREM-1 levels (P > 0.05). No remarkable difference was found between the noninfected group and the control group (P > 0.05). No remarkable difference in TREM-1 level was found before and after intravitreal antibiotics (P > 0.05). TREM-1 was selectively highly expressed on the surface of cell membrane of neutrophils and monocytes/macrophages infiltrated in vitreous and uveal of the PTBE group. The area under the ROC curve (AUC) was 0.79 (>0.75), with a medium diagnostic efficiency. The sensitivity and specificity of sTREM-1 to differentiate PTBE from the noninfected intraocular condition were 62.50% and 86.25% separately. A cutoff value >524.50 pg/mL for sTREM-1 was predicted to be PTBE. Vitreous sTREM-1 levels in PTBE group were positively correlated with PES (r = 0.428, P < 0.05). However, sTREM-1 levels and FBCVA did not significantly correlate with one another (P > 0.05).

Conclusions: The sTREM-1 was a promising diagnostic biomarker of PTBE, especially CN-PTBE. Vitreous sTREM-1 levels were linked with intraocular inflammation levels and severity of PTBE.

Endophthalmitis is a vision-threatening complication of intraocular infection, which can cause inflammation, retinal harm, and, finally, loss of visual acuity and blindness.^1,2 Post-traumatic bacterial endophthalmitis (PTBE), an intraocular suppurative inflammatory illness brought on by open globe injuries, is the most typical form of infectious endophthalmitis.³ In addition to the virulence and pathogenicity of the causative agents, the rapid provision of suitable therapy also affects the clinical prognosis.⁴ The most crucial element in preventing potentially fatal visual consequences lies in early detection, which allows for timely management. The clinician's experience and microbiological analysis are essential in making a diagnosis. However, extensive ocular tissue damage and noninfectious ocular conditions caused by trauma can be difficult to distinguish from mild and early PTBE. 

Microbial culture of ocular fluid is the “gold standard” for the diagnosis of PTBE, but it is time-consuming and lacks sensitivity evaluation in certain location.⁵ Some patients with highly suspected infectious endophthalmitis have negative microbial culture results, further complicating the evaluation.⁶ Earlier studies have found that culture-positive rate was low, ranging from 31.7% to 38.1%,⁷ which might be related to prior antibiotic medication that may impede microbial development during culture. On the other hand, infection with a lower load of microorganisms will also lead to a low positive rate.⁸ PCR testing has obvious advantages compared with microbial culture, which is not only fast but also has a high detection rate. However, PCR requires specific primers and might have a high false-positive rate because of nonspecific amplification.⁹ A previous study using next-generation sequencing to identify individuals with infectious endophthalmitis found that nearly 50% of culture-negative (CN) samples had pathogenetic DNA. However, next-generation sequencing is an advanced technology and has a turnaround cycle of almost five days with complex processes and high cost.⁸ Recently, rapid antigen detection has been implemented for COVID-19 screening, which needs only 15 minutes, but only selected pathogens can be detected. 

A biomarker can be highly valuable in rapid and early diagnosis of infectious endophthalmitis, particularly in culture-negative cases and may facilitate skipping time-consuming diagnostic processes and the administration of antibiotics. However, some biological indicators have been investigated in an effort to enhance the diagnosis process, such as procalcitonin, but with unsatisfactory outcomes.^10–12 Therefore it is urgent to find more sensitive and specific candidate molecules. 

The idea of using the soluble triggering receptors expressed on myeloid cells (sTREM-1) to discriminate between sepsis and systemic inflammatory response syndrome has been discussed.¹³ TREM-1 is a component of the immunoglobulin superfamily, and exposure to bacteria increases its expression on myeloid cells.¹⁴ Neutrophils and monocytes with elevated TREM-1 expression are infiltrated into bacterially contaminated human tissues. TREM-1 is, however, barely expressed in samples from individuals with noninfectious inflammatory disorders.^15,16 Activation of the TREM-1 pathway enhance the inflammatory response and TREM-1 is a potential target for treating infectious diseases such as sepsis.¹⁷ TREM-1 is also released from the membrane of excited myeloid cells and becomes a soluble form in bodily fluids. It has been suggested that the marker sTREM-1 may be helpful for identifying infectious from non-infectious disorders^18,19 and can also evaluate the severity and predict the prognosis.^16,20 Because nothing is known about their usage in PTBE, especially for culture-negative PTBE (CN-PTBE), we hypothesize that vitreous sTREM-1 measurements can be used to distinguish between infected and non-infected vitreous humor (VH), and TREM-1 could be a potential biomarker of PTBE. 

Methods

Subjects

Before enrolling, 94 patients or their family members gave written informed consent that was authorized by the institutional review board for the study (Ethics Approval No:2019-K068). Thirty-two patients with a diagnosis of PTBE were included in the study from the Ophthalmology Department in Affiliated Hospital of Nantong University between June 2020 and July 2022 (Jiang Su, China). The clinical diagnostic criteria of PTBE were recorded as follows: (1) history of open globe injuries; (2) severe eye pain, photophobia, tearing, sharp decline in vision, or even complete loss; (3) eyelid edema, conjunctiva congestion, edema, corneal opacity and hypopyon; (4) B ultrasound of eyes suggested vitreous opacity; and (5) culture positive for bacteria supporting the diagnosis of PTBE, although negative results cannot exclude the possibility of PTBE. Intravitreal antibiotic (IVA) was used for suspected or early endophthalmitis cases or as an initial treatment before pars plana vitrectomy (PPV) surgery. The characteristics of patients with suspected endophthalmitis and who need emergency PPV are summarized as follows: (1) presence of intraocular foreign body (IOFB), vitreous hemorrhage, or retinal detachment; (2) rapid deterioration of visual acuity to hand movement or below, and rapid deterioration of the disease; (3) no improvement or worsening of symptoms after IVA; and (4) B ultrasound scanning showed large amounts of vitreous turbidity or abscess. 

For the noninfected group, 62 patients in need of vitrectomy or evisceration for traumatic noninfectious diseases, such as IOFB, retinal detachment, or vitreous hemorrhage, were enrolled. For all open globe injuries, infection was ruled out by intraocular fluid smear and stain, microbial culture, and clinical findings during hospitalization or at admission. 

For the control group, 21 samples were collected from subjects deceased from various causes such as car accident without eye trauma, cerebral hemorrhage, and kidney failure and with recorded consent to donate eyeballs. All group patients were excluded if they presented with a record of uveitis, a record of ocular surgery or trauma, and a history of ocular infection. 

Peyman Endophthalmitis Scores

Peyman endophthalmitis scores (PES) were determined by eyelid and conjunctival swelling, corneal opacity, anterior chamber fibrin or hypopyon, vitreous opacity, and visibility of the fundus and were graded from 0, 1, 2, to 3, representing absence, mild, moderate, and severe reaction, respectively. 

Sample Collection

VH (0.3–0.5 mL) was extracted with a 1 mL syringe before IVA. VH was also acquired during PPV (500 mm Hg, 5000 r/min) before administration of a balanced salt solution. Uveal or retina tissues were obtained during evisceration for eye atrophy, panophthalmitis, and more. Centrifugation was used to separate the supernatants of VH, which was then kept at −80°C until testing. 

STREM-1 Assays

The concentration of VH sTREM-1 was determined by using commercially available human ELISA kits (QIAOYI, Shantou, China) as per the manufacturer's instructions. In short, the 96-well plates received 100 µL of samples, which were then incubated. To prevent background readings caused by NADH or NADPH present in the specimen, a sample blank was preserved. All samples and sTREM-1 standards were examined simultaneously and carried out twice. Using a UV-Vis spectrophotometer, the samples were read at A450 after 30 minutes of incubation in the dark. Using interpolation, the sample sTREM-1 concentrations were calculated from points produced by the standard curve. The test is finished in roughly three to four hours or less. 

Hematoxylin-Eosin Staining (HE)

After evisceration, the uveal tract, retina, and abscess were collected and fixed in 4% formalin for histopathological testing. The dehydration, wax dip, embedding, sectioning, and HE staining conducted by the Institute of Ophthalmology in Affiliated Hospital of Nantong University. 

Immunohistochemistry (IHC)

Enucleated eyeballs or their contents' paraffin slices were deparaffinized and hydrated by being exposed to xylene, classed alcohol, and water. Paraffin sections were treated with microwave-based antigen retrieval (pH = 6.0) in 10 mM citrate buffer, blocked endogenous peroxidase for five minutes with 3% H₂O₂, and then incubated with a primary mouse monoclonal antibody against TREM-1 (1:500; Abcam, Cambridge, MA, USA) at 4°C overnight before being exposed to HRP (Protein Bio, Fuzhou, China) for 20 minutes at ambient temperature. All sections were then stained with hematoxylin for two to three minutes and then treated with DAB Chromogen (ZS-Biotech, Lahore, Pakistan) for three minutes each. The sections were then dehydrated and cleared with ethanol and xylene. As a substitute for primary antibodies, PBS was used. 

Statistical Analysis

SPSS 24.0 was used to analyze the data. Data were expressed as the median with the interquartile range (IQR) or as the mean with the SD. By using the Kolmogorov-Smirnov test, the continuous data's normality was evaluated. In the case of numerical data, all groups were compared with each other by non-parametric Kruskal-Wallis test (or the Mann-Whitney U test, if appropriate). Difference in TREM-1 concentrations before and after IVA were assessed using the Wilcoxon test. LogMAR was used to translate the Snellen visual acuity. LogMAR were scaled as follows: 2.00 for finger counting, 2.30 for hand movement, 2.60 for light perception (LP), and 2.9 for no LP.⁴ At the six-month follow-up, the final best-corrected visual acuity (FBCVA) was obtained. Using Spearman's correlation test, the relationships between sTREM-1 and biological or clinical characteristics were evaluated. By receiver operating characteristic (ROC) curve calculation, sTREM-1's diagnostic performance was assessed. Using the SPSS 24.0, ROC curve was plotted. Youden's indices were computed after the sensitivity and specificity for various TREM-1 cutoff values were assessed. The best cutoff point was chosen using the Youden's index's maximum value as a criterion. Statistical significance was considered as P < 0.05. 

Results

Characters of the Patients

Our study collected 32 patients (32 eyes) clinically identified as PTBE, of which 21 (65.6%) patients were culture positive (CP) for bacteria by routine microbiological work-up and 11 (34.4%) patients were culture negative (CN) (Tables 1, 2). Ten patients (10 eyes) in PTBE accepted the initial IVA, four of which then received PPV, one of which then received evisceration, five of which were cured. One (one eye) patient in PTBE accepted the evisceration directly. Twenty-one patients (21 eyes) in PTBE accepted the emergency PPV directly. 

Table 1.

 

Summary of Demographic Data of the Patients Included in the Study

 

Table 2.

 

Cultured Microorganisms in PTBE Group

 

The average year in non-infected group was 53.56 ±11.65 years and included 54 males. The control group included 11 donors (21 eyes). The mean age of the control group was 60.81 ± 12.90 years and included nine males. 

Vitreous sTREM-1 Concentration in the PTBE, NonInfected, and Control Groups

The concentration of vitreous sTREM-1 in PTBE group (median with IQR 720.34, 212.56–1439.90 pg/mL) was higher than that in non-infected group (160.85, 104.56–288.41 pg/mL) (P < 0.05) and control group (102.13, 46.52–218.38 pg/mL) (P < 0.05). No remarkable difference was found between noninfected and control group (P > 0.05) (Fig. 1A). 

Figure 1.

 

STREM-1 values for all research participants' vitreous samples that were examined. Mann-Whitney U test between two groups. Kruskal–Wallis H test among three or more groups. Wilcoxon test before and after IVA *P < 0.05; **P < 0.01; ns, P > 0.05. Bar: Median with IQR.

No remarkable difference was found between CP group (median with IQR:601.38, 218.34-1471.31 pg/mL) and CN group (779.20, 239.20-1221.67 pg/mL) (P > 0.05) in sTREM-1 levels. TREM-1 levels in CP and CN group were all higher than that in noninfected (160.85, 104.56–288.41 pg/mL) (P < 0.05) and control group (102.13, 46.52-218.38 pg/mL) (P < 0.01), as shown in Figure 1B. 

There were also no expression level differences in TREM-1 in Gram-positive (1253.73, 212.10-1532.51 pg/mL) and gram-negative (444.85, 218.34-1784.29 pg/mL) traumatic endophthalmitis (Fig. 1C). In culture-positive endophthalmitis, Pseudomonas aeruginosa endophthalmitis showed the highest concentration of TREM-1 expression level, which was 5406.51 pg/mL. 

Four patients (four eyes) accepted emergency vitrectomy and one patient (one eyes) accepted the evisceration after initial IVA. Their sTREM-1 levels before and after IVA were supplemented as follows: 779.20 versus 3163.15, 576.88 versus 2369.42, 221.23 versus 276.51, 601.38 versus 335.57, and 138.05 versus 941.36 (pg/mL) respectively. No remarkable difference in TREM-1 level was found before and after administration of IVA (Fig. 1D). 

HE and IHC

All uveal and pus in vitreous were removed, sectioned, and stained with hematoxylin and eosin for visualization of general immune cell. Inflammation of the vitreous and uveal were observed in the PTBE group (Figs. 2, 3) and the noninfected group (Fig. 4). More severe inflammation infiltration was observed in PTBE group (Figs. 2A, 3A) compared with noninfected group (Fig. 4A). TREM-1 was positively expressed on the surface of inflammatory cell membrane in the PTBE group (Figs. 2B, 2C, 3B, 3C), whereas TREM-1 was not expressed in the noninfected group (Figs. 4B, 4C). The retina and choroid were largely intact in the control group (Fig. 5). 

Figure 2.

 

HE and IHC staining of pus in CP-PTBE group. (A) Inflammatory cells (many neutrophils and a few monocytes/macrophages) were infiltrated in the pus of the vitreous. (B, C) TREM-1 was positively expressed on the surface of inflammatory cell membrane. (A, B) 200X; (C) 600X.

Figure 3.

 

Choroid HE and IHC staining in CP-PTBE group. (A) Inflammatory cells (many neutrophils and a few monocytes/macrophages) were infiltrated in choroid of PTBE patients. (B, C) TREM-1 was positively expressed on the surface of inflammatory cell membrane. (A, B) 200X; (C) 600X.

Figure 4.

 

Choroid HE and IHC staining in non-infected group. (A) Few inflammatory cells were infiltrated in choroid of non-infected group. (B) TREM-1 was not expressed on the surface of inflammatory cell membrane. (A, B) 200X.

Figure 5.

 

Choroid HE and IHC staining in control group. (A) The structure of the choroid and retina were normal. (B) TREM-1 was not expressed in normal choroid and retina tissues. (A, B) 200X.

Figure 6.

 

ROC curves of sTREM-1 for the diagnosis of PTBE.

ROC Curves of sTREM-1 for the Diagnosis of PTBE

The diagnostic score of STREM-1 was determined by the ROC curve (Fig. 6). The AUC of STREM-1 was 0.79 (95% CI, 0.69–0.88), and the cutoff value was 524.50 pg/mL when the Youden index was at its maximum. The corresponding sensitivity and specificity were 62.50% and 86.75% (P < 0.01). 

Correlation Analysis of sTREM-1 Concentrations With Endophthalmitis Scores in Patients With PTBE

Correlation analysis (Fig. 7) showed that sTREM-1 concentration was positively correlated with PES (r = 0.467, P < 0.05). 

Figure 7.

 

Correlation analysis of sTREM-1 concentration with PES in patients with PTBE.

Figure 8.

 

Correlation analysis of sTREM-1 concentration with FBCVA in patients with PTBE.

Correlation Analysis of sTREM-1 Concentration With FBCVA in Patients With PTBE

No evidence (Fig. 8) of a statistical relationship between sTREM-1 levels and FBCVA was found (r = 0.182, P > 0.05). 

Discussion

Discrimination of the infectious or noninfectious intraocular condition after open globe injury remains a clinical challenge, but the emergence of TREM-1 may help to solve this dilemma. The results of this study initially indicated that sTREM-1 could be a promising biomarker of PTBE, especially CN-PTBE, even though IVA has been administrated, which could guide clinicians in diagnosis and treatment. Our study also discovered that sTREM-1 was positively linked with ocular inflammatory status and a biomarker for the severity of PTBE. STREM-1 levels were detected by ELISA promptly in our study (three to four hours or less) and provided valuable information long before the microbial culture result was back. The newest SimpleStep ELISA kit offers results within 90 minutes without compromising analytical performance.²¹ Moreover, the assay is affordable and could be used in most clinical laboratories without sophisticated equipment and reagents. 

The ROC curve is often used to determine a cutoff that can distinguish between genuine infection and noninfected controls. With a VH sTREM-1 concentration of 524.50 (pg/mL) as the cutoff number for PTBE diagnosis, the sensitivity and specificity are 62.50% and 86.75% (AUC 0.75). Qin et al.²² discovered that blood sTREM-1 had a good sensitivity of 0.85 (95% CI, 0.76–0.91) and a mild specificity of 0.79 (95% CI, 0.70–0.86) to distinguish sepsis from systemic inflammatory response syndrome. Huang et al.²³ found that the determination of pleural sTREM-1 may improve the ability of clinicians to differentiate bacterial pleural effusion from those with other causes with a sensitivity of 86% and a specificity of 93%. A previous meta-analysis found that sTREM-1 is a valuable biomarker of bacterial lung infections in patients in the intensive care unit with a sensitivity of 87% and a specificity of 79% (AUC 0.91).²⁴ However, the low sensitivity of our study could be related to the sampling. Endophthalmitis cases caused by IOFBs accounted for the majority of our study. All endophthalmitis patients with IOFB received emergency PPV within three hours or less after admission to our hospital. Infection and inflammation could be limited at the posterior pole or inferior retinal surface where the IOFBs were, but the position of the vitreous aspiration was in the anterior segment and midaxial vitreous, which could cause low readings of the measurement. Even with unsatisfactory sensitivity, sTREM-1 could promptly provide valuable auxiliary information for doctors faced with ambiguous endophthalmitis with a good specificity of 86.75%. 

CN-PTBE is frequently masked when bacteria are low in number and prior antibiotic administration is applied.⁶ Infection with these nonculturable microorganisms would also lead to CN-PTBE.²⁵ The present study showed that sTREM-1 could be a biomarker of CN-PTBE and useful for distinguishing CN-PTBE from noninfected uveitis after open globe injury. The rationale is that inflammatory storms respond anyway if bacteria exist even though the bacteria is not detectable because of a low number or is unculturable. TREM-1 may act as an amplifier of inflammatory response to mediate the formation of inflammatory storm. Moreover, our study also found that administration of IVA did not affect the expression of sTREM-1. Inflammation may be prolonged, even though the bacteria has been cleared by antibiotics. Toxins or metabolites produced by bacteria may also contribute to inflammation. So sTREM-1 could also have diagnostic value after IVA, which is better than microbial culture, but the sample size of present cases is insufficient to establish a solid conclusion in this regard. 

PES was used to judge the inflammation condition and severity of experimental rabbit endophthalmitis.²⁶ STREM-1 is a marker of the activation of TREM-1 pathway, and the increased expression of sTREM-1 indicates excessive inflammatory response in the eye and the severity of the disease.²⁷ The present study discovered that sTREM-1 was positively linked with PES and severity of PTBE. As we know, the prognosis of Pseudomonas aeruginosa endophthalmitis is extremely poor, and the evisceration rate is quite high.²⁸ In our study, one case of P. aeruginosa traumatic endophthalmitis showed the highest concentration of sTREM-1 levels, indicating severe inflammation in ocular circumstances. This patient received evisceration directly. Serum sTREM-1 was a biological indicator for diagnosis of mycoplasma pneumonia and was positively correlated with disease severity.²⁹ Serum sTREM-1 was linked with periodontitis and disease severity in rheumatoid arthritis.³⁰ These reports suggest that sTREM-1 can be used to predict infectious diseases, as well as to judge the severity, which was consistent with our results. 

Silva et al.³¹ discovered sTREM-1 levels in serum were elevated in patients with novel coronavirus-infected pneumonia and could be used to judge prognosis. However, our data were unable to find a connection of sTREM-1 levels to FBCVA. We know that many variables, such as the course of disease, the virulence of microorganisms, the location of trauma, inflammation, injury degree of trauma, and retinal detachment, could influence FBCVA of PTBE.^4,32,33 

Recently, a TREM-1 inhibitor (Nangibotide) is projected to be the first specific treatment for sepsis shock after achieving ground-breaking outcomes in phase IIb clinical studies.³⁴ Our study found that TREM-1 is highly expressed on inflammatory cells infiltrated in vitreous and choroid of PTBE patients, which provides a potential therapeutic target for the treatment of PTBE. Given the poor visual results found in PTBE administered with antibiotics alone or in combination with corticosteroids, anti-TREM-1 immunomodulation with the addition of antibiotic therapy may serve as a novel strategy for protecting visual acuity. 

Conclusions

To sum up, our study showed that VH sTREM-1 levels could be helpful in distinguishing between PTBE (especially CN-PTBE) and noninfectious uveitis. Our study also discovered sTREM-1 was positively linked with ocular inflammation activity and a biomarker for severity of PTBE. Antibiotic therapy might be warranted if a patient is clinically diagnosed with endophthalmitis with a high vitreous sTREM-1 level but a negative microbial culture. With the discovery of a particular target like TREM-1, novel treatment strategies may be of interest in the future. Given that endophthalmitis patients have a high risk of poor prognosis, more in-depth and multicenter research is needed to evaluate the diagnostic value of sTREM-1. 

Acknowledgments

Supported by the Science and Technology Project of Nantong Municipality (no. MS12020033). 

Disclosure: Q. Tang, None; M. He, None; S. Zhang, None; J. Zhang, None; L. Yang, None; H. Shi, None