Pythium insidiosum keratitis reported in China, raising the alertness to this fungus-like infection: a case series

A 45-year-old female of Han nationality presented to a cirneal clinic in August, 2017 with a history of pain, redness, and decreased vision in her right eye 1 week after being exposed to river water. Corneal scrapings and confocal microscopy in vivo were performed instantly. A mass of hyphae was found in the 10% KOH wet mount stained with lactophenol blue and examined via confocal microscopy. Fungal keratitis was identified, and right corneal keratectomy was performed. Empirical antifungal and antibacterial therapy was initiated including topical and systemic fluconazole, levofloxacin, and cefminox sodium. After treatment of 2 weeks, the ulcer and symptoms did not improve and the patient was admitted to our hospital. Conjunctival congestion persisted, and a central corneal ulcer with a diameter of around 6 mm that reaches deep into the stromal layer can be seen (Fig. 1A, B). Subepithelial and superficial stromal opacities with dot-like and tentacle-like infiltrates accompanied this. Intracameral fluconazole injection was used to conduct a lamellar keratoplasty. Amphotericin B was administered immediately after the surgery. The first day post-surgery, hyperemia and a thin exudation membrane in the anterior chamber were discernible (Fig. 1C, D). Four days after the keratoplasty, full-thickness large central infiltrate with hypopyon was observed in the right eye (Fig. 1E, F). Given the increasing infiltrate with hypopyon and ineffectiveness of antifungal therapy, the right eye was enucleated. Microbial culture for corneal tissue revealed fungus-like organism showing long sparsely septate hyaline hyphae (Fig. 1G, H). The organism was further identified as P. insidiosum by ribosomal RNA (rRNA) gene sequencing with panfungal primers (ITS1/ITS4), which matched 99.23% with the P. insidiosum strain (GU137348.1). Then, the patient was adjusted with combined antibacterial treatment consisting of linezolid and azithromycin, and no recurrence of infection was observed on follow-up.

A 51-year-old female of Han nationality was referred to our hospital in September 2018 with complications of pain, redness, and hyperemia in her right eye after entry of some cigarette ash 1 week ago. Corneal scraping was performed, and Gram staining, KOH preparation, and cultures were negative. Antifungal and antibacterial treatment was initiated including levofloxacin eye drops, cefminox sodium, and voriconazole. After 2 weeks of outpatient treatment, the patient was hospitalized. A grayish-white ulcer was observed in the central of bitamporal cornea measuring 4 × 6 mm. Inflammatory infiltrates with feathered margins and hypopyon with a depth of 2 mm were seen, suggestive of a fungal infection. The cornea showed dense central stromal opacity surrounded by a reticular pattern of subepithelial and superficial stromal infiltration (Fig. 2). Then penetrating keratoplasty was performed. Exudation was observed in anterior chamber on 2 days following surgery, and intracameral fluconazole injection was performed. However, the infiltrates extended progressively, and were unresponsive to any treatment. Therefore, by day 28 post-exposure, an enucleation was performed to remove infected tissue and relieve pain. One week later, a small amount of mycelial growth was observed within the corneal fragment on the potato dextrose agar (PDA) plate. Subculture in brain–heart infusion resulted in the rapid growth of a large mycelium at 35 °C. We have attempted to identify this mycelium by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF-MS) but failed. There was no reference spectrum in the Bruker Filamentous Fungi databases despite the high quality of the protein spectrum. The mycelium was sent for internal transcribed spacer (ITS) rRNA gene sequencing analysis and unambiguously identified as P. insidiosum.. Three obvious protein peaks of the strain were found by MALDI-TOF-MS, which are 2094.01, 4834.62, and 9682.51 (× 103 m/z), respectively (Fig. 3). Then, the reference spectrum of P. insidiosum was added to the in-house database according to Bruker MALDI-TOF-MS standard process to create a library. Therefore, the identification of P. insidiosum could be facilitated using MALDI-TOF-MS.

A 55-year-old male of Han nationality presented with irritation, pain, and hyposia in the left eye for 3 days after facial washing with contaminated river water. He went to a local ophthalmic clinic and was diagnosed with viral keratitis. After 4 days of antiviral therapy, the symptoms were not improved, so the patient was admitted to our hospital. Slit-lamp examination showed conjunctival hypertrophy and infiltrated growth into cornea about 2 mm from the nasal limbus. In the left eye, microvascular tissue hypertrophy of about 2.5 mm was seen across the corneal limbus, and hyperemia grayish-white infiltrate of cornea with a diameter of about 5 mm was observed (Fig. 4). Direct microscopy of corneal scrapings stained with Gram and KOH preparation was negative for organisms. The patient was prescribed empiric fortified topical and systemic antibiotics, including ornidazole, tobramycin, vancomycin, natamycin, and fluconazole. On the third day after hospitalization, symptom improvement was not noted. Excision of pterygium and therapeutic penetrating keratoplasty were performed in the left eye. Cultures of his corneal tissue for bacteria, fungus, and Acanthamoeba were negative. Because there was evidence of increased keratoneuritis, antiamebic therapy (chlorhexidine) was initiated and voriconazole was added. Twelve days after the surgery, corneal opacity was rescraped and infiltration extended deeply into the anterior chamber. A second penetrating keratoplasty, virtually limbus to limbus, and intracameral amphotericin B injection were performed sequentially. However, 10 days after the second operation, the infection still spread to the adjacent sclera and progressed to endophthalmitis. Enucleation eventually had to be done. Subsequently, the corneal cultures growing on PDA plate were identified as P. insidiosum by MALDI-TOF-MS, confirming the diagnosis of pythiosis.