Effective diagnosis by real-time PCR of herpes simplex diffuse endotheliitis that is similar in appearance to fungal keratitis: case series

Purpose Herpes simplex diffuse endotheliitis with accompanying feathery infiltration is difficult to diagnose due to corneal findings that are similar to fungal keratitis. This case series reports on the effectiveness of using real-time polymerase chain reaction (PCR) to diagnose herpes simplex diffuse endotheliitis that is similar in appearance to fungal keratitis. Methods After extracting corneal smear sample DNA, samples were then applied to two independent PCR assays, a qualitative multiplex 24-pathogen strip PCR assay, and a quantitative real-time PCR assay of herpes simplex virus type 1 (HSV-1). Results All 3 cases showed ciliary injection, feathery infiltration in the corneal stroma and hypopyon, which are corneal findings similar to that observed for fungal keratitis. Retrocorneal plaques, which showed clear boundaries between the corneal endothelial surfaces and retrocorneal plaques in anterior segment optical coherence tomography, were observed in 2 out of 3 cases. Corneal scraping was performed in all cases, followed by initiation of antifungal treatment. However, real-time PCR of the corneal scraping detected 6.0 × 106, 1.0 × 105 and 5.0 × 105 copies/μg glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of HSV-1 DNA per each microgram of the samples. Fungi were not cultured in any of the cases. After switching the medication from antifungal to antiviral, the feathery corneal infiltration was cured with only mild scarring. Conclusions Real-time PCR was an effective tool in diagnosing HSV diffuse endotheliitis with feathery infiltration. Topical corticosteroids in conjunction with oral and topical antivirals were an effective treatment.

Turicella otitidis: a rare agent causing microbial keratitis

A 10-year-old boy treated for alkali injury with multiple interventions presented with a perforated corneal ulcer with clinically suspected bacterial aetiology. Cornea scraping and tissue adhesive application were planned. During surgery, an eyelash was found embedded at the perforated site. Gram staining of corneal scraping revealed the presence of Gram-positive bacilli on the first day which later was identified as Turicella otitidis with culture followed by VITEK V.2.0 (Biomerieux) identification. The bacterium was found to be sensitive to amikacin, ciprofloxacin, cefazolin, gatifloxacin, moxifloxacin, ofloxacin and vancomycin antibiotics as per Clinical and Laboratory Standards Institute guidelines. Coryneform bacteria is a rare cause of keratitis, and this is the first reported case of microbial keratitis caused by one of the rare corynebacterium species T. otitidis to the best of our knowledge. Literature search does not reveal any specific ocular features typical to this organism. This case supports the growing evidence for pathogenicity of T. otitidis in ocular samples. This study demonstrates the utility of VITEK for the identification of rare pathogen and may facilitate the use of certain antibiotics in the treatment regimen of T. otitidis infections.

Fusarium infection complicating rheumatic keratitis that acutely progressed to endophthalmitis during regular infusion of tocilizumab: a case report

Background Filamentous fungi are ubiquitous in plants, water, and soil. The predominant fungi that infect the human cornea include Fusarium and Aspergillus species. The onset of fungal endophthalmitis is indolent, and typically takes weeks to months to develop after corneal infection. We report a case of Fusarium infection complicating rheumatic keratitis that acutely progressed to endophthalmitis during intravenous tocilizumab therapy. Case presentation A 65-year-old female patient was referred to our department due to pain and decreased vision in her left eye. Slit-lamp examination showed a white focus on the upper peripheral cornea, hypopyon, anterior chamber fibrin formation, marked ciliary hyperemia, and whole corneal epithelial defects. As the corneal scraping smear was positive for filamentous fungi and Fusarium species were detected by aqueous humor polymerase chain reaction, anti-fungal therapy was started. Although the initial response to anti-fungal therapy was good, we observed corneal infiltration, worsening hypopyon, and vitreous opacity after tocilizumab infusion. Given that the infection continued to progress despite conservative therapy, we performed penetrating keratoplasty combined with vitrectomy. After removal of the white focus beneath the intraocular lens, a temporary corneal prosthesis was mounted and the dense vitreous opacity was removed. Finally, a frozen donor graft was sutured in place. The corneal infiltration, hypopyon, and vitreous opacity all disappeared after the operation. Conclusion The rapid progression of Fusarium keratitis to endophthalmitis in a patient who was receiving a regular infusion of tocilizumab demonstrates that ocular condition should be closely monitored during systemic tocilizumab administration due to increased risk of infection.

Effective Diagnosis by Real-Time PCR of Herpes Simplex Diffuse Endotheliitis That is Similar in Appearance to Fungal Keratitis: Case Series

PURPOSE: Herpes simplex diffuse endotheliitis with accompanying feathery infiltration is difficult to diagnose due to corneal findings that are similar to fungal keratitis. This case series reports on the effectiveness of using real-time polymerase chain reaction (PCR) to diagnose herpes simplex diffuse endotheliitis that is similar in appearance to fungal keratitis.METHODS: After extracting corneal smear sample DNA, samples were then applied to two independent PCR assays, a qualitative multiplex 24-pathogen strip PCR assay, and a quantitative real-time PCR assay of herpes simplex virus type 1 (HSV-1).RESULTS: All 3 cases showed ciliary injection, feathery infiltration in the corneal stroma and hypopyon, which are corneal findings similar to that observed for fungal keratitis. Retrocorneal plaques, which showed clear boundaries between the corneal endothelial surfaces and retrocorneal plaques in anterior segment optical coherence tomography, were observed in 2 out of 3 cases. Corneal scraping was performed in all cases, followed by initiation of antifungal treatment. However, real-time PCR of the corneal scraping detected 6.0 x 106, 1.0 x 105 and 5.0 x 105 copies/μg glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of HSV-1 DNA per each microgram of the samples. Fungi were not cultured in any of the cases. After switching the medication from antifungal to antiviral, the feathery corneal infiltration was cured with only mild scarring.CONCLUSIONS: Real-time PCR was an effective tool in diagnosing HSV diffuse endotheliitis with feathery infiltration. Topical corticosteroids in conjunction with oral and topical antivirals were an effective treatment.

Metagenome Techniques for Detection of Pathogens Causing Ocular Infection

Metagenomic analysis is the comprehensive study of DNA using clinical specimens of organisms including bacteria, fungi, and viruses. In this study, we investigated the efficacy of metagenomic analysis for diagnosing ocular infections, including 11 keratitis cases, four iridocyclitis cases, and one endophthalmitis case. Corneal scraping, aqueous humor, and vitreous humor, were collected respectively. Ocular specimens were used for bacterial and fungal culture, and PCR for detecting viral DNA. Shotgun metagenomic sequencing for 150 bases of single end was performed by Illumina MiSeq® System. Sequence was retrieved from the database at NCBI using a MegaBLAST search. Since Propionibacterium spp. are commensal bacteria found at the ocular surface, they were excluded from analysis. Six cases (37.5%) were positive for culture or PCR. Metagenome techniques revealed that 9 cases (56.3%) included genomes of organisms that were considered pathogenic in specimens. Five cases (31.3%) possessed genomes of organisms like themselves that were detected by culture and PCR. Six cases (37.5%) were negative for culture, PCR, and metagenome analysis. Moreover, viral pathogens (HSV-1, 2 cases; and VZV, 1 case) were detected by only metagenome analysis. Metagenome analysis using an ocular sample can detect microbial genome comprehensively, and viral pathogens, which were not detected by conventional examination.

Clinical Spectrum of Corneal Epithelial Microsporidiosis

This case series aims to highlight three different clinical variants of microbiologically proven epithelial microsporidiosis, and their customised management wherein the current series, patients with suspected epithelial microsporidiosis were subjected to corneal scraping to confirm the diagnosis. Different microbiologically proven clinical variants were observed and documented. Three clinical variants of epithelial microsporidiosis were noted. The first was of 45-years-old male patient, presented with only raised punctate lesions, and responded to topical lubricants only. The second variant, 28-years-old male patient, had anterior chamber reaction (flare and cells), Keratic Precipitates and descemets folds. This variant also responded with topical lubricants over a period of one month. The third variant, 32-years-old male patient at presentation, had typical epithelial raised punctate corneal lesions, however at one week subepithelial infiltrates appeared. These lesions responded to topical steroids. The current series highlights three different clinical variants of microbiologically proven epithelial microsporidiosis and their customised management.

A ten-year report of microbial keratitis in pediatric population under five years in a tertiary eye center

Purpose To report characteristics of microbial keratitis in pediatric patients under five years. Methods Patients with infectious keratitis under the age of 5 years were included in this retrospective cross-sectional study for ten years. All patients were admitted and corneal scraping was performed in 81 children. Fortified empiric antibiotic eye drops including cefazolin (50 mg/cc) and amikacin (20 mg/cc) were started and the antibiotic regimen was continued or changed according to culture results. In the case of fungal keratitis, topical voriconazole (10 mg/cc) or natamycin (50 mg/cc) and topical chloramphenicol (5 mg/cc) were started. A tectonic procedure was done when corneal thinning or perforation was present. Results Ninety-Three Patients between 1 to 60 months with a mean age of 33 ± 18 months old with corneal ulcer were included in the study. The most common risk factor was trauma (40.9%) followed by contact lens use (8.6%). Cultures were negative for microbial growth in 28 (30.1%) patients. The most common pathogens were S. epidermidis (10.8%) and P. aeruginosa (10.8%). Fluoroquinolone antibiotics (ciprofloxacin; 93.8% sensitivity) were the most potent antibiotic against bacterial pathogens. Forty-one patients underwent tectonic procedures, which the most common ones were cyanoacrylate glue 18.3% followed by keratoplasty 16.1%. Conclusion This study emphasizes the role of trauma as the primary cause and S. epidermidis as the most frequent microorganism in pediatric keratitis; according to antibiogram results and poor cooperation of patients under five years, monotherapy with fluoroquinolones could be a good regimen in small non-central lesions without thinning.

PCR and culture for diagnosis of Acanthamoeba keratitis

Background/AimsAcanthamoeba keratitis (AK) is a rare but sight-threatening infection. Molecular diagnosis of corneal scraping has improved the diagnosis of AK. Different molecular targets and conditions have been used in diagnosis thus far. In this study, we prospectively compared the performance of five PCR assays on corneal samples for the diagnosis of AK.Methods1217 corneal scraping samples were obtained from patients, for whom an AK was suspected. Sample processing involved both molecular diagnostics and culture. Acanthamoeba PCR assays detected different regions of the Acanthamoeba nuclear small-subunit rRNA gene: three final point PCR assays using Nelson, ACARNA and JDP1–JDP2 pairs of primers, and two real-time PCR assays using Acant primer-probe. Human DNA and internal control were co-amplified in the real-time PCR assay to ensure scraping quality and the absence of inhibitors. In the absence of a gold standard, the performance of each test was evaluated using latent class analysis. Genotypes of Acanthamoeba isolates were also characterised.ResultsEstimated prevalence of AK was 1.32%. The sensitivity of Acanthamoeba diagnostic PCRs (73.3% to 86.7%) did not differ significantly from that of culture (66.7%), or according to the target sequence or the technology. Sensitivity could be increased to 93.8% or 100% by combining two or three assays, respectively. PCR specificity (99.3% to 100%) differed between the assays. T4 was the predominant Acanthamoeba genotype (84.6%).ConclusionsCulture and a single PCR assay could lead to misdiagnosing AK. A combination of different PCR assays and improved sample quality could increase diagnosis sensitivity.

Cyphellophora sp. Isolated from a Corneal Ulcer in the Human Eye

Cyphellophora is a black yeast-like fungus with most of the strains being isolated from soil and plants. It tends to cause sooty blotch and flyspeck disease in plants. In humans, it is known to cause superficial skin and nail infections. This report highlights the case of a patient who initially presented with a small corneal abrasion which rapidly progressed into a corneal ulcer after the patient did not respond to the initial conventional treatment. The laboratory results from the corneal scraping found it to be Cyphellophora sp.

12-year analysis of incidence, microbiological profiles and in vitro antimicrobial susceptibility of infectious keratitis: the Nottingham Infectious Keratitis Study

Background/aimsTo examine the incidence, causative microorganisms and in vitro antimicrobial susceptibility and resistance profiles of infectious keratitis (IK) in Nottingham, UK.MethodsA retrospective study of all patients who were diagnosed with IK and underwent corneal scraping between July 2007 and October 2019 (a 12-year period) at a UK tertiary referral centre. Relevant data, including demographic factors, microbiological profiles and in vitro antibiotic susceptibility of IK, were analysed.ResultsThe estimated incidence of IK was 34.7 per 100 000 people/year. Of the 1333 corneal scrapes, 502 (37.7%) were culture-positive and 572 causative microorganisms were identified. Sixty (4.5%) cases were of polymicrobial origin (caused by ≥2 different microorganisms). Gram-positive bacteria (308, 53.8%) were most commonly isolated, followed by Gram-negative bacteria (223, 39.0%), acanthamoeba (24, 4.2%) and fungi (17, 3.0%). Pseudomonas aeruginosa (135, 23.6%) was the single most common organism isolated. There was a significant increase in Moraxella spp (p<0.001) and significant decrease in Klebsiella spp (p=0.004) over time. The in vitro susceptibilities of Gram-positive and Gram-negative bacteria to cephalosporin, fluoroquinolone and aminoglycoside were 100.0% and 81.3%, 91.9% and 98.1%, and 95.2% and 98.3%, respectively. An increase in resistance against penicillin was observed in Gram-positive (from 3.5% to 12.7%; p=0.005) and Gram-negative bacteria (from 52.6% to 65.4%; p=0.22).ConclusionIK represents a relatively common and persistent burden in the UK and the reported incidence is likely underestimated. Current broad-spectrum antimicrobial treatment provides a good coverage for IK, although challenged by some level of antimicrobial resistance and polymicrobial infection.