The Therapeutic Effect of Corneal Transplantation for Refractory Pseudomonas Aeruginosa Corneal Ulcer

Purpose To observe the treatment outcome of corneal transplantation for advanced medically uncontrolled culture-proven pseudomonas aeruginosa corneal ulcer.Design Retrospective analysisSubjects and methods 26 patients (eyes) with refractory culture-positive pseudomonas aeruginosa corneal ulcer who failed to respond to drug therapy and underwent consecutive corneal transplant procedures in a hospital (2008.1-2018.8). Etiology, medical history, clinical features, surgical type, vision, recurrence, complications and treatment were recorded, and the relationship between postoperative recovery and selection of surgical method was analyzed.Results Of the 26 patients with pseudomonas aeruginosa corneal ulcer, 9 (34.6%) received penetrating keratoplasty (PKP) and 17 (65.4%) received lamellar keratoplasty (LKP). 22 patients (84.6%) obtained a successful outcome through one corneal transplantation. Of the 9 patients who received PKP, 1 patient having graft rejection 6 months after surgery (endothelial type) obtained successful outcome through adequate drug treatment., while 1 case received success by graft repair combined with amniotic membrane transplantation on the 5 months postoperatively for fungal corneal graft ulcer. In the 17 patients underwent LKP, 2 received a second successful lamellar corneal transplantation for corneal graft melting 2 months after the �rst surgery. In all the 26 patients, the corneal infection was effectively brought under control by corneal transplantation, and none of them had recurrent ulcers during at least 6months' follow-up. The visual acuity was signi�cantly improved at the last follow-up compared with that before surgery. The postoperative visual acuity of patients underwent LKP was better than that of those who underwent PKP ( p =0.018).Conclusions Corneal transplantation can effectively treat refractory pseudomonas aeruginosa corneal ulcer worsening despite adequate medical treatment and improve eyesight. Compared with PKP, LKP can be the main surgical method to treat refractory pseudomonas aeruginosa corneal ulcer.


Introduction
Bacterial corneal ulcer, as a kind of microbial keratitis, is still the main cause of eye diseases and blindness in the world, especially in developing countries [1][2][3][4][5][6].Among gram negative bacteria, pseudomonas aeruginosa is the most common [7].It has been reported that pseudomonas aeruginosa is the most important pathogen in refractory bacterial corneal ulcer [8].Pseudomonas aeruginosa corneal ulcer, a serious blinding eye disease, with rapid onset and development, whose treatment is very di cult, can lead to corneal perforation, endophthalmitis, acute vision loss even the prolapsus of eye viscera.Therefore, timely and effective treatment is very necessary.Current treatments include drug therapy, amnion transplantation, conjunctival ap occlusion, penetrating keratoplasty, and lamellar keratoplasty.A large number of studies have reported the treatment of pseudomonas aeruginosa corneal ulcer with drug therapy, amniotic membrane transplantation, conjunctival ap covering and so on.However, few studies have reported corneal transplantation for the treatment of pseudomonas aeruginosa corneal ulcer since 1964 [8][9][10][11].Corneal transplantation can not only completely remove bacteria, but also improve the transparency of the cornea and signi cantly increase visual acuity [12][13][14][15].This study discussed the clinical characteristics, surgical indications, surgical effects and complications of refractory pseudomonas aeruginosa corneal ulcer treated with corneal transplantation in the hospital.

Subjects And Methods
This retrospective case analysis was approved by the Ethics Committee of the institute and complied with the Helsinki Declaration.We retrospectively analyzed the medical records of patients diagnosed with refractory pseudomonas aeruginosa corneal ulcer and received corneal transplantation in the hospital from January 2008 to August 2018.We analyzed the demographics of these patients, ulcer characteristics, results of smear and culture tests, and antibiotic sensitivity, methods of corneal transplantation, and postoperative recovery.The effects were evaluated by infection control, visual acuity recovery, and complications.
Pseudomonas aeruginosa corneal ulcer was diagnosed using the following methods: (1) clinical slit-lamp and anterior segment optical coherence tomography (AS-OCT) (OPTOVUE) examination revealed the modality and depth of the corneal ulcer respectively, (2) corneal scrapings at admission showed a positive pseudomonas aeruginosa culture result, (3) confocal microscopy examination (HRT3-RCM) imaged no fungal hyphae or acanthamoeba cysts to exclude fungal and amoeba infection respectively.Of all the 26 patients (eyes), 6 eyes (23.1%) with corneal perforation and 2 eyes (7.7%) with imminent corneal perforation were not given confocal microscopy examination.Each patient underwent B-scan ultrasounds in order to exclude endophthalmitis.De nition of refractory pseudomonas aeruginosa corneal ulcer: (1) drug treatment is ineffective; (2) the ulcer is larger than 6mm in diameter, or deeper than 1/2 corneal thickness, or smaller but located within 3mm of the optic axis (3) corneal perforation or imminent corneal perforation.
According to the operating regulations of our hospital, all patients were given corneal scrapings, bacterial culture and drug sensitivity test before taking any antibiotics.Speci c methods: according to standard cultivation of microorganism separation procedures [16,17], ophthalmologist scraped both the base and edge of the ulcer with sterile blades under aseptic conditions after instillation of 4% Xylocaine, then inoculated the samples in blood and chocolate culture media, which also subjected Gram's stain and 10% potassium hydroxide wet microscopy, and meanwhile swabbed them to broth for enriched culture with sterile swabs to identify bacteria and fungi species and test antibiotic susceptibility.
Drug treatment: all patients received two or three broad spectrum antibiotic eye drops such as levo oxacin eye drops, 10% ceftazidine eye drops tobramycin eye drops (Alcon, Fort Worth, TX, USA) once every half an hour, along with 1g ceftazidine intravenous drip 3 times a day, 0.5g levo oxacin intravenous drip once a day, and o oxacin eye ointment (Santen, Osaka, Japan) at night.Then adjust the medication according to the drug sensitivity result.Surgical treatment should be considered if timely and adequate medication fails or the ulcer worsens for two weeks.
Penetrating corneal transplantation for those with Descemet membrane invasion or even perforation, otherwise with lamellar corneal transplantation.Penetrating corneal transplantation procedure: with manual dissection technique, the abnormal corneal tissue was cut out by a trephine with a diameter 0.25 mm larger than the ulcer and the DX-preserved donor corneal graft was sutured into the graft bed with 10/0 nylon thread.Lamellar keratoplasty procedure: the necrotic corneal tissue was continuously exfoliated layer by layer until the left portion was clear.The glycerine-preserved donor graft was xed onto the implant bed with 10/0 nylon thread by intermittent suture.
Postoperative treatment: Three to ve days after surgery, all eyes were treated with tobradex eye ointment 4 times daily, subsequently with 0.1% or 0.02% uorometholone eye drops 4 times a day according to the control of in ammation 5-7 days after surgery, which was decreased gradually within two weeks.1% Cyclosporin A eye drops 3 times daily and tobradex eye ointment once each night were given when the epithelium repaired.1% Cyclosporin A eye drops and 0.02% uorometholone eye drops were given 2 times a day, besides with tobradex eye ointment once per week three months after surgery.
Statistical analysis: SPSS 17.0 (SPSS, Inc, Chicago, Illinois, USA) was used for data analysis.A value of p< 0.05 was considered statistically signi cant.
Results of culture and drug sensitivity test of corneal scraping: there were a large number of pus cells in each corneal spatula in 26 patients, and gram negative bacilli was detected in 14 patients, with a positive rate of 53.8%.Pseudomonas aeruginosa was grown in culture in all 26 patients.Drug susceptibility test results: pseudomonas aeruginosa cultured from corneal smear of 26 patients, 24 cases were sensitive to tobramycin, 24 sensitive to ceftazidime, 23 sensitive to levo oxacin, and for the three kinds of drugs, 19 cases were sensitive to all of them, 6 cases were sensitive to two kinds of them, and 1 case was only sensitive to cephalosporin.Among all pseudomonas aeruginosa of the 26 patients, 15 were resistant to ceftriaxone, whose minimum inhibitory concentration (MIC) was more than 32, and no resistance was found in 3 patients.
Postoperative recovery: In all the 26 patients, the corneal infection was effectively brought under control by corneal transplantation (Figure 2, Figure 3), and none of them had recurrent ulcers during at least 6months' follow-up.For all the 26 patients, 22 patients (84.6%) obtained successful outcome through one corneal transplantation.9 patients received penetrating keratoplasty and 17 received lamellar keratoplasty.Of the 9 patients who received PKP, 1 patient having graft rejection 6 months after surgery (endothelial type) obtained successful outcome through adequate drug treatment, while 1 case received success by graft repair combined with amniotic membrane transplantation on the 5 months postoperatively for fungal corneal graft ulcer.In the 17 patients underwent LKP, 2 received a second successful lamellar corneal transplantation for corneal graft melting 2 months after the rst surgery.The best corrected visual acuity of all patients at the last follow-up was signi cantly improved compared with that before surgery (Table 2).The postoperative visual acuity of patients underwent lamellar keratoplasty was better than that of those who underwent penetrating keratoplasty (Figure 4).

Discussion
The pseudomonas aeruginosa, as a kind of conditional pathogenic bacteria and cause of hospital acquired infection, can induce multiple organ damage, such as respiratory tract, urinary system, nervous system, eyes, ears, nose , throat and so on, and even sepsis heavier and illness, treatment more di cult.In the eye, pseudomonas aeruginosa may cause keratitis, corneal perforation, sharp vision loss and even endophthalmitis [18], which may be related to its virulence [19].Therefore, timely and effective treatment is particularly important.Current treatments include drug therapy, amnion transplantation, conjunctival ap occlusion, penetrating keratoplasty, and lamellar keratoplasty.It has been reported that although frequent drugs can control in ammation to a certain extent, they are toxic to corneal epithelium, and vision was affected for corneal scarring [20].Both amniotic membrane transplantation and conjunctival ap occlusion promoted epithelial healing, but the bacteria could not be completely removed, and neither visual acuity was improved [21][22][23][24][25]. Corneal transplantation, thoroughly removing bacteria, could improve the transparency of the cornea and signi cantly increased vision.Compared with the penetrating corneal transplantation, lamellar corneal transplantation has the advantages of more donors, smaller rejection, less complications, and a more ideal visual effect [26][27][28].In this study, of the 26 patients, the corneal infection was effectively controlled by corneal transplantation.Twenty-two patients (84.6%) achieved therapeutic success after one corneal transplantation, consistent with the results of the relevant studies [29].Of the 9 patients underwent PKP, 1 patient had graft rejection 6 months after surgery, while 1 case underwent fungal corneal graft ulcer on the 5 months postoperatively.In the 17 patients underwent LKP, 2 had corneal graft melting 2 months after the rst surgery.The best corrected visual acuity of all patients was signi cantly improved at the last follow-up compared with that before surgery, and lamellar keratoplasty was more conducive to the improvement of postoperative visual acuity than penetrating keratoplasty.
It has been reported that bacterial corneal ulcer was mostly caused by corneal trauma ocular surface disease and wearing corneal contact lens [30,31].Our study showed that, the most common cause of pseudomonas aeruginosa corneal ulcer was corneal trauma, which may be a result of the occupations of our patients, as most of them were agricultural or other out-doors workers.
As the gold standard for the diagnosis of bacterial corneal ulcer, corneal spatula and bacterial culture are indispensable.In our study, there were a large number of pus cells in each corneal sample in 26 patients, and gram negative bacilli was detected in 14 patients, with a positive rate of 53.8%, which was consistent with related reports [32,33].Drug susceptibility test results: pseudomonas aeruginosa cultured from corneal smear of 26 patients, 24 cases were sensitive to tobramycin, 24 sensitive to ceftazidime, 23 sensitive to levo oxacin, and for the three kinds of drugs, 19 cases were sensitive to all of them, 6 cases were sensitive to two kinds of them, and 1 case was only sensitive to cephalosporin.The result was consistent with relevant reports [32,34].Among all pseudomonas aeruginosa of the 26 patients, 15 were resistant to ceftriaxone, whose minimum inhibitory concentration (MIC) was more than 32, and no resistance was found in 3 patients.

Conclusions
In conclusion, corneal transplantation can effectively treat refractory pseudomonas aeruginosa corneal ulcer and improve eyesight.Lamellar keratoplasty has the advantages of more donors, smaller rejection, less complications, and a more ideal visual effect, and can be the main surgical method to treat refractory pseudocycosis corneal ulcer.The de ciency of this study is that the number of cases is so small that analysis of related factors for corneal transplantation in the treatment of refractory pseudomonas aeruginosa corneal ulcer was not statistically signi cant, and further clinical data should be collected in the future to better guide future clinical work.

Figures
Figures

Figure 1 Demographic 2 LKP for pseudomonas aeruginosa corneal ulcer Figure 3 PKP for pseudomonas aeruginosa corneal ulcer Figure 4
Figure 1