Background
Corneal transplantation is the most commonly performed tissue or organ transplant worldwide. Penetrating keratoplasty (PKP), a procedure consisting of full-thickness replacement of the cornea, has been the dominant procedure for more than half a century. In recent years, lamellar keratoplasty techniques have replaced PKP for anterior and posterior corneal pathologies. These include deep anterior lamellar keratoplasty (DALK), Descemet stripping automated endothelial keratoplasty (DSAEK)/Descemet membrane endothelial keratoplasty (DMEK).
Post-keratoplasty infectious keratitis is a devastating complication that may severely affect corneal graft survival rate and visual outcomes [
1]. Fungal keratitis post-keratoplasty is actually more common than bacterial or viral keratitis [
2,
3]. A study by the Eye Bank Association of America encompassing 4 years (2007 − 2010) of activity, reported a cumulative frequency of post-keratoplasty infection rate of 0.026% for fungal and bacterial agents together, with a higher rate for fungal isolates (63%). The most common fungal infection, particularly in endothelial keratoplasty (EK), are from the
Candida genus, with the majority caused by either
Candida albicans or
Candida parapsilosis [
4].
Early-onset
Candida infection after keratoplasty commonly presents as a white infiltrate that may not be associated with significant inflammation. The dense infiltration can result in vision loss especially in patients with deep interface infection after EK, which can also spread to the anterior chamber resulting in endophthalmitis. Early diagnosis of post-keratoplasty
Candida infection is very important for prognosis. Confocal microscopy is a useful tool to confirm the diagnosis of the infectious microorganism especially in patients with deep corneal infections. It has a good diagnostic accuracy especially for amoebic keratitis and fungal keratitis [
5,
6]. Treatment of fungal keratitis is a clinical challenge. Intrastromal and interface antifungal therapy has been effective in arresting the progression of fungal infection for late-onset lamellar interface infectious keratitis [
7].
In this study, we report six cases of Candida keratitis after keratoplasty (3 post PKP and 3 following DSAEK). We documented the clinical and confocal microscopy features, as well as the outcomes of the management.
Methods
This study was performed in accordance with the Declaration of Helsinki. The study was approved by the ethics committee of Tianjin Medical University Eye Hospital. Written informed consent was obtained from the patients. Six patients with laboratory diagnosis of Candida keratitis after keratoplasty were examined at Tianjin Medical University Eye Hospital from 2018 to 2021. Culture of corneal scraping (4 cases) and corneal biopsy (2 cases) were performed to establish the diagnosis.
Specimens for corneal scraping cytological analysis were obtained using a surgical blade and the sample was fixed in methanol, then stained with Giemsa. Specimens for culture were also obtained using a surgical blade. In 2 post-DSAEK patients with interface infection, a corneal biopsy was taken in the operating theater under topical anesthesia. A corneal incision at the peripheral edge of the infiltrate was performed, taking care to avoid the visual axis. Specimens from corneal scrapings or a tissue biopsy were inoculated on Sabouraud dextrose agar (at 22–25 °C) and blood agar (at 37 °C). Isolates were identified by MALDI-TOF-MS system (Bruker, Germany).
In vivo confocal microscopy scanning with the HRTIII-RCM (Heidelberg Engineering GmbH, Dossenheim, Germany) was performed on all patients. The morphology, distribution and depth of Candida spp. in the cornea was observed using 800x magnification.
All patients were immediately treated with topical voriconazole (VCZ) 1% and topical natamycin (NTM) 5% every hour, when the result of in vivo confocal microscopy examination or corneal scraping for cytological analysis was positive. Patients with mid/deep stromal keratitis or interface infection were treated with a combination of oral VCZ (400 mg, two times a day). Two post-PKP patients with mid/deep stromal keratitis were additionally treated with intrastromal VCZ injection. Intrastromal injection of VCZ (0.05 mg/0.1mL) with a 30-g needle around the active peripheral lesion was performed. The amount of drug injected intrastromally was about 0.1mL. And two post-DSAEK patients with interface infection were additionally treated with interface VCZ irrigation. Interface irrigation with VCZ (0.05 mg/0.1mL) was also performed with a 30-g needle. The amount of drug was about 0.1mL.
Results
The clinical data of the patients in this study was outlined in Table
1. The 6 patients included three males and three females, between 32 and 89 years old. Three patients underwent PKP and three underwent DSAEK. After surgery, all the patients were given topical corticosteroids to reduce the risk of graft rejection. The time to onset of infection after surgery ranged from 1 to 9 months. The cytological analysis of the corneal scrapings was positive for
Candida in four of six patients. The culture of the corneal scraping (4 cases)/ biopsy (2 cases) for
Candida spp. was positive in all patients.
Candida albicans was the most common species isolated (4 cases), followed by
Candida dubliniensis (1 case) and
Candida lusitaniae (1 case).
Table 1
Clinical data of patients with Candida keratitis after keratoplasty
1,89,M | OS | DSAEK | 5 months | White infiltrate in the mid anterior stroma | + | Candida dubliniensis | + | VCZ (topical) NTM (topical) | 1 month | CF | 20/200 |
2,65,M | OS | PKP | 3 months | White infiltrate in the mid stroma | + | Candida albicans | + | VCZ (topical + intrastromal) NTM (topical) | 45 days | 20/160 | 20/80 |
3, 56,M | OD | DSAEK | 2 months | Graft-host interface infiltration | - | Candida albicans | + | VCZ (topical + interface irrigation + oral) NTM (topical) | 2 months | 20/160 | 20/80 |
4,62 F | OS | DSAEK | 1 month | Graft-host interface infiltration | - | Candida albicans | - | VCZ (topical + interface irrigation + oral) NTM (topical) Remove graft | 2 months | CF | HM |
5,32,F | OD | PKP | 3 months | White infiltrate in the deep stroma around the loose suture | + | Candida albicans | + | VCZ (topical + intrastromal + oral) NTM (topical) | 2 months | 20/100 | 20/80 |
6, 67,F | OS | PKP | 9 months | White infiltrate in the anterior stroma | + | Candida lusitaniae | + | VCZ (topical) NTM (topical) | 2 months | CF | 20/200 |
In vivo confocal microscopy examination was positive in five of six patients (3 post-PKP and 2 post-DSAEK). Fungal spores were identified in all five patients. One patient was positive for both spores and pseudohyphae structures. The fungal spores were hyperreflective round deposits, about 2–5 μm in diameter, distributed in clusters. No obvious inflammatory cell infiltration was observed around the fungal spores. Pseudohyphae were characterized by high-density striate structures with clusters of round spore images around them.
After therapy, infection in five of six patients was completely resolved within 45–60 days. Only one post-DSAEK patient (case 4) with severe infection had the corneal endothelium graft removed. Final uncorrected visual acuity (UCVA) ranged from HM to 20/80.
Discussion
Post-keratoplasty infectious keratitis is a well-known clinical challenge. The incidence of infectious keratitis after keratoplasty varies widely, ranging from 0.02 to 11.9% depending on the specific type of transplant (PKP, DALK, or EK) [
8‐
10]. Early diagnosis and treatment of post-keratoplasty
Candida infection is important, especially in cases of deep stromal or interface infiltrates. In this study, six patients who developed
Candida keratitis postoperatively were collected. The clinical features, diagnostic testing including in vivo confocal microscopy, and treatment outcomes were analyzed.
Topical corticosteroids are usually administered following keratoplasty to reduce the risk of graft rejection [
8]. The use of corticosteroids, however, is a double-edged sword. A 15-year review of clinical outcomes of 21 cases of
Candida keratitis in a Canadian eye center suggested that the use of topical corticosteroids was a common risk factor [
11]. In our study, topical steroids were routinely given to all patients following keratoplasty. The local immunosuppressive effect of steroids may be an important risk factor for
Candida infection. Other well-known risk factors for infectious keratitis include persistent epithelial defects and loose sutures [
12]. Two of our patients (one post-PKP, the other post-DSAEK) had a history of delayed epithelialization. In our series of patients, severe ocular surface disease (SJS) and tear dysfunction were also contributing risk factors. One of our post-PKP patients (case 5) had a loose suture, a well-known risk factor. Loose sutures cause epithelial defects which can be contaminated by environmental and ocular surface commensals.
Post keratoplasty
Candida keratitis is typically asymptomatic associated with minimal inflammation. The patients may present with a quiet white eye, with mild vision loss as the only presenting symptom, especially in early-onset interface infection after DSAEK. Most
Candida interface keratitis develop several weeks to several months after EK surgery [
13]. In our series, the interval from surgery to onset of infection was 1 to 9 months. Since the sequestered location of the infection in the deep stroma or interface along with the reduced virulence of the
Candida organism, is associated with an asymptomatic presentation, the diagnosis is often delayed. Furthermore, the impaired access to the deep location of the organism, contributes to the challenges of microbiological testing and the clinical management of these cases.
In this restrictive setting of cases of
Candida spp. infection, in vivo confocal microscopy can provide an important diagnostic tool by identifying hyperreflective round structures consistent with
Candida. In our series, confocal microscopy was positive in five of six patients. Our imaging of
Candida spores was similar to previous reports [
14,
15]. The size of the spores was about 2–5 μm in diameter, distributed in clusters. Pseudohyphae were identified by their characteristic high-density striate structure. We also noted a lack of inflammatory cell infiltration around spores and pseudohyphae. Three different species of
Candida were identified in our patients. Although the size and appearance of these 3 species of spores were similar, the pseudohyphae were only found in the patient with
Candida dubliniensis keratitis (case 1). In vitro formation of pseudohyphae is related to the species of
Candida, the type of culture plates and the temperature. Pseudohyphae is also an indication of the virulence of the
Candida spp. In the identification test between
Candida albicans and
Candida dubliniensis on staib agar,
Candida dubliniensis produced rough colonies, peudohyphae and chlamydospores. Although the
Candida albicans produced pseudohyphae after prolonged growth [
16].
However, in our opinion, we were unable to accurately identify the genus of Candida from in vivo confocal microscope images. Morphological features of fungal colonies and spores require up to 7 days for fungi to grow and sporulate in culture. In contrast, the high-resolution imaging modality of in vivo confocal microscopy provides immediate visualisation of fungi within living cornea.
In general, cultures of corneal scraping constitute the standard methodology to diagnose fungal infection [
17]. However, in our study, due to the deep location of the infection in two post-DSAEK patients who had developed graft-host interface infections, corneal scrapings would not be helpful in identifying the culprit organism. For these 2 cases, we performed interface corneal biopsy. Firstly, a corneal incision at the peripheral edge of the infiltrate was performed. Then we use the forceps to get the specimens. The results showed
Candida albicans infection. Corneal biopsy is the definitive procedure to establish a diagnosis in progressive keratitis, especially if corneal scraping yields negative results [
18]. Notwithstanding this technique carries some risks, including intraoperative corneal perforation as well as corneal scarring.
Standard topical antifungal therapy with NTM 5% and VCZ 1%, has limited efficacy for deeper interface infection due to the poor penetration [
19]. Intrastromal injection and interface irrigation of VCZ is a preferred adjunctive treatment approach in the management of deep
Candida keratitis. This therapy can provide maximum drug load at the site of the infection, especially at the graft–host interface, with the aim of salvaging the graft and avoiding PKP [
20]. In a previous study, the success rate of intrastromal VCZ in treating recalcitrant fungal keratitis (ulcer size > 2 mm, a depth > 50% of stroma, and not responding to topical NTM therapy for two weeks) was 95% [
21]. In addition, interface irrigation with antifungal agents has reportedly been effective in clearing interface infection, but carries a risk of graft dislocation [
9]. This adjunctive treatment was effective in our patients, in which the graft was successfully salvaged. In case 4, however, with severe interface infection after DSAEK, the graft was dislocated when we performed interface irrigation. Therefore, we had to remove the graft and plan for a PKP. One of the main limitations of our study was the small sample size. Other limitations included a lack of culture results for donor cornea rim. So the risk of the contamination from the donor tissue could not be analyzed. Occurrence of fungal keratitis in the recipient of the donor’s fellow eye may be indicative of donor related source of infection. Of course, during the surgery of fungal keratitis, we should notice sufficient graft diameter, always interrupted sutures, no double running suture, and anterior chamber irrigation.
In conclusion, Candida spp. demonstrates characteristic morphological features on in vivo confocal microscopy. In cases of deep stromal or interface infiltrates following keratoplasty, which may be inaccessible by corneal scraping, the use of confocal microscopy demonstrating white deposits with hyperreflective round structures, may provide important support of the possibility of Candida keratitis. In these cases, intrastromal and interface injection of antifungal agents can be a safe and useful adjunct to standard topical and systemic antifungal therapy for the management of fungal keratitis. Early diagnosis and treatment may lead to a better prognosis for patients with Candida keratitis after keratoplasty.
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