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Pythium insidiosumkeratitis confirmed by DNA sequence analysis
  1. PAUL R BADENOCH,
  2. DOUGLAS J COSTER
  1. Department of Ophthalmology, Flinders University of South Australia and Flinders Medical Centre, Bedford Park, South Australia
  2. Department of Clinical Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, South Australia
  3. Department of Anatomical Pathology, Flinders Medical Centre, Bedford Park
  4. South Australia
  5. CRC for Tropical Plant Pathology, University of Queensland, St. Lucia
  6. Queensland
  1. BRUCE L WETHERALL,
  2. HELEN T BRETTIG
  1. Department of Ophthalmology, Flinders University of South Australia and Flinders Medical Centre, Bedford Park, South Australia
  2. Department of Clinical Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, South Australia
  3. Department of Anatomical Pathology, Flinders Medical Centre, Bedford Park
  4. South Australia
  5. CRC for Tropical Plant Pathology, University of Queensland, St. Lucia
  6. Queensland
  1. MICHAEL A ROZENBILDS
  1. Department of Ophthalmology, Flinders University of South Australia and Flinders Medical Centre, Bedford Park, South Australia
  2. Department of Clinical Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, South Australia
  3. Department of Anatomical Pathology, Flinders Medical Centre, Bedford Park
  4. South Australia
  5. CRC for Tropical Plant Pathology, University of Queensland, St. Lucia
  6. Queensland
  1. ANDRÉ DRENTH,
  2. GABRIELE WAGELS
  1. Department of Ophthalmology, Flinders University of South Australia and Flinders Medical Centre, Bedford Park, South Australia
  2. Department of Clinical Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, South Australia
  3. Department of Anatomical Pathology, Flinders Medical Centre, Bedford Park
  4. South Australia
  5. CRC for Tropical Plant Pathology, University of Queensland, St. Lucia
  6. Queensland
  1. Dr Paul Badenoch, Department of Ophthalmology, Flinders Medical Centre, South Australia 5042, Australia

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Editor,—Pythium insidiosum is an unusual but serious ocular pathogen. Although the organism grows as a mycelium in tissue, it is not a member of the fungal kingdom and its identification can be a challenge for a routine laboratory. We report a case of Pythiumkeratitis in which the organism was confirmed by nucleic acid sequencing.

CASE REPORT

A 32 year old man was referred from Kuala Lumpur having suffered with intractable keratitis of the left eye for 4 weeks. He gave a history of diabetes, disposable contact lens wear, and swimming in the Kelang River. Routine microbiological investigations had been negative. At presentation to Flinders Medical Centre, he was on topical antibacterial, antifungal, and antiamoebic medication. He had a large epithelial defect, a deep stromal infiltrate approaching the limbus, and hypopyon (Fig 1, top). His visual acuity was hand movements and there was considerable pain. The drops were stopped and corneal scrapings were taken. Gram and Giemsa stains were negative. A biopsy was performed the following day and hyphae were observed in sections. A filamentous organism appeared in cultures of the original scrapings. There was no response to continued antifungal treatment and a penetrating graft was performed 4 days after the biopsy. Postoperatively, the patient received oral itraconazole and topical natamycin. Prednisolone phosphate drops were introduced 10 days later. Twice in the first 3 weeks after surgery the patient returned to theatre for an anterior chamber washout of proliferative material invading from the peripheral cornea (Fig 1, bottom). Hyphae were seen in these specimens, but cultures were negative. The patient returned to Malaysia and, 7 months postoperatively, had a clear graft, useful vision in the eye, and no recurrence of infection.

Figure 1

Pythium insidiosum keratitis. Top, the patient at presentation. Bottom, proliferative material in the anterior chamber after corneal transplantation.

The histopathology revealed a florid keratitis with necrotic stroma and degenerate neutrophils and monocytes. Massive numbers of hyphae were seen in silver-stained sections, particularly in the anterior stroma (Fig 2, top). Hyphae were also observed penetrating Descemet's membrane. The organisms were not stained by the periodic acid-Schiff (PAS) method, also commonly used in suspected fungal infection, giving these sections the appearance of Swiss cheese (Fig 2, bottom).

Figure 2

Histopathology of the excised cornea (×625). Top, silver stain. Bottom, PAS. The organisms have not stained with PAS; their presence is indicated by the spaces in the section.

Colonies grew rapidly on the primary fungal medium (plain Sabouraud's agar, 28°C). They were white with a yellowish tinge, unusually flat, and difficult to cut and separate from the agar. Few septae and no spores were seen. The isolate was sent to a reference laboratory under suspicion of being a zygomycete. However, no further taxonomic clues were induced by standard measures so a molecular approach was employed. The methods have been described.1 Initially, a 510 base segment of the 18S ribosomal RNA gene was amplified (universal primers NS1 and NS2)2 and sequenced (Model 373A DNA sequencer, Applied Biosystems Inc). A search of databases (GenBank, EMBL) revealed homology to oomycetes. The isolate was then incubated for 48 hours in water containing autoclaved grass. Motile zoospores were observed, indicative of P insidiosum. Other characteristics consistent with this identification were colony morphology, optimal temperature of growth (35°C), hyphal diameter (4–6 mm), intercalary swellings in viable hyphae, vesicles at the end of spore discharge tubes, and spores germinating by means of germ tubes.3

To confirm the identification, the internal transcribed spacer (ITS) region defined by primers TW81 and AB28 (incorporating ITS1, the 5.8S gene and ITS2) was amplified. The 900 base pair product was sequenced by a direct double stranded DNA cycle method using primers 1–6 as detailed1 except for the substitution of primer 3 with 3c (5'-GCGACTTCGGTTAGGACATT). The sequences were checked between complementary strands and shown to be 99.0% homologous withP insidiosum reference strain CBS777.84 using CLUSTAL V software4 (CA Lévesque, personal communication). In comparison, the sequence was ∼95% homologous with other Pythium species and ∼90% homologous with other genera of oomycetes (BLAST database, NIH, Bethesda, MD, USA).

COMMENT

P insidiosum is an aquatic, filamentous organism that produces heterokont, biflagellate zoospores, placing it as an oomycete in the kingdom Chromista. The only other human pathogen in this kingdom isRhinosporidium seeberi, the agent of rhinosporidiosis. Oomycetes have a diploid genome and a cell wall consisting of cellulosic compounds and glycan, features which distinguish them from the fungal groups of the kingdom Fungi. The identification of an oomycete in our patient can explain the PAS result and the lack of response to antifungals, there being no chitin or ergosterol in the cell wall, respectively. In addition to our genetic approach to confirming the identification, serological tests have been developed in Bangkok5 and at the Centers for Disease Control, Atlanta.

Oomycetes play an important part in the decomposition and recycling of decaying matter. Some species are parasitic on crops and fish. In mammals, P insidiosum causes a granulomatous disease (“swamp cancer”) in several species including horses and dogs. A number of cases of human pythiosis have been reported, mostly subcutaneous infections and arteritis in thalassaemic patients in farming communities of South East Asia.6 With respect to the eye, P insidiosum has been responsible for periorbital infections in Australia7 and the USA8 and corneal ulcers in Thailand,6Haiti,9 and New Zealand.10 Some patients have had no other medical history. Contact lens wear, exposure to river water, and diabetes were possible predisposing factors in our patient.

No cases of Pythium keratitis have been cured medically. A boy with facial pythiosis was cured without surgery by a 1 year course of terbinafine and itraconazole.8 For the cornea, however, it would be difficult to imagine a successful outcome given the destructive nature of the organism, the slow response if any to most antimicrobial agents, the probable delay in identification, and the need to prevent further tissue invasion. Transplantation may be the best option.

The introduction of corticosteroids in the management of fungal keratitis and, presumably, Pythiuminfection, must be approached with great caution. We gave prednisolone in the hope of reducing inflammation in the grafted cornea without encouraging a recurrence of the disease. It was fortunate that, apparently, no infectious elements had been left in the eye.

References

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