Lasiodiplodia theobromae keratitis: A rare tropical fungal keratitis in a non-tropical climate

Purpose We present the clinical and histopathological findings of a geographically unique Lasiodiplodia theobromae fungal keratitis case in North Carolina. L. theobromae is a rare cause of fungal keratitis, and all but one of the 51 previously reported cases have occurred in patients living in the tropics. Observations A man in his early 50s developed L. theobromae keratitis after being struck in the left eye by a piece of debris while using a flexible-cord weed trimmer. Intracapsular lensectomy and penetrating keratoplasty were necessary when initial antimicrobial therapy was ineffective. The best-corrected visual acuity was 20/40 four years postoperatively. Conclusions and Importance Our patient is only the second example of L. theobromae keratitis in a patient living in a sub-tropical climate and the first case in the U.S.A. outside of Florida. Additional in-vitro antibiotic sensitivity testing and documentation of more clinical cases are needed to define the optimal therapy for Lasiodiplodia theobromae keratitis.


Introduction
Lasiodiplodia theobromae, also known as Botryodiplodia theobromae, is a dematiaceous (pigmented) fungus common in the tropics, 1 where it is a frequent wound parasite on plants such as bananas. 2 In the southern United States, this fungus is a common cause of wood stain, 3 and it is also associated with plant rot in cotton, citrus fruit, Honey Dew melons, and strawberries. 4L. theobromae is a rare cause of severe keratitis, sometimes requiring penetrating keratoplasty. 5][8] We present the successful treatment of a man from North Carolina with corneal infection by this unusual fungus.Our patient represents, to our knowledge, the first individual in the U.S.A. outside of Florida who has developed L. theobromae keratitis.

Case report
A man in his early 50s was struck in the left eye by a piece of debris while using a flexible-cord weed trimmer.An outside ophthalmologist evaluated him the next day for ocular discomfort and foreign body sensation.Visual inspection disclosed an epithelial infiltrate with putative organic material at its base in the left paracentral cornea.The foreign material was extracted, and the eye was patched after applying 3.5 mg/gm neomycin/10,000 U/mg polymyxin B sulfate/0.1 % dexamethasone (Maxitrol®) ointment.Topical ciprofloxacin was started two days due to the persistence of the epithelial infiltrate and inflammation.There was a slight blurring of his vision, and the best corrected visual acuity in the left eye was 20/40.Intraocular pressure was 8 mmHg O.D. and 13 mmHg O.S. Therapy with ciprofloxacin was ineffective, and 15 days after injury there was stromal opacification superocentrally that appeared to penetrate the deep stroma.There was a small area of white deposit on the endothelium, along with one or two cells in the anterior chamber.
Over the next month, the patient experienced waxing and waning left eye pain and a scratching sensation.He was treated with atropine and various topical antimicrobials/antifungals, including ampicillin, ciprofloxacin, chloramphenicol, and natamycin.His care during this month also included two anterior chamber taps, from which cultures were negative.At the end of the second month following his injury, there was swelling and erythema of the left eyelids, increasing injection of the conjunctiva, and a hypopyon.He was referred to Duke University for further evaluation and treatment.
Approximately two months after the original injury best corrected visual acuity was 20/20 O.D. and 20/200 O.S. Intraocular pressure was 12 mmHg O.D. and 16 mmHg O.S. Slit lamp biomicroscopy found superficial punctate keratopathy, a superficial scar with deep corneal infiltrate, a plaque on the endothelium, and a small hypopyon.There was no epithelial defect at this time, and the vitreous and retina appeared unremarkable.The clinical impression was deep fungal keratitis.An anterior chamber tap and biopsy of the endothelial plaque were done that day.Gram stain of the aqueous sample showed rare leukocytes, but no organisms.Potassium hydroxide (KOH) preparation of the sample was negative, as were fungal, bacterial, and acid-fast bacilli cultures.Cytological examination of the endothelial plaque was negative for both organisms and malignancy.The patient was started on topical natamycin and amphotericin B, and oral ketoconazole, but the endothelial plaque persisted.
Two months later, there were keratic precipitates and filaments extending from the posterior cornea into the anterior chamber.Despite the persistence of the endothelial plaque and presumed fungal extension, visual acuity improved during this time and stabilized at 20/60 O. S. A fourth anterior chamber tap was performed four months following the original corneal injury.KOH/calcofluor white microscopic examination was negative, and the remainder of the fluid was cultured dropwise without streaking onto six agar plates and one agar slant.Three of the drops grew Lasiodiplodia theobromae, and the other four drops showed no growth.
Pharmacological management was changed to topical amphotericin B, natamycin, and flucytosine, and subconjunctival miconazole based on the identification of the fungus as L. theobromae.The endothelial plaque persisted, and at five months post-injury, an infiltrate was extending from the posterior cornea to the anterior lens capsule, with an accompanying decrease in visual acuity to 20/400 O.S.The patient was started on oral flucytosine, along with his ocular medications; visual acuity slowly improved, and the infection appeared to also improve and stabilize but not resolve.Examination of the left eye revealed 1 to 2+ injection of the conjunctiva, a central corneal scar with several endothelial deposits and adjacent keratic precipitates (Fig. 1A and B), a 1 mm fluffy deposit on the lens capsule with filaments extending into the anterior chamber, and several smaller deposits on the lens capsule.The lens was otherwise clear.At six months following the injury, additional lenticular involvement prompted a penetrating keratoplasty, intracapsular cataract extraction, and anterior vitrectomy.Postoperatively, the patient received topical amphotericin B and flucytosine, and oral itraconazole.No recurrence of the infection occurred, and the antifungals were discontinued after three months.
Pathological examination of the corneal button disclosed granulomatous inflammation near the center of the cornea in a region where Descemet's membrane was discontinuous (Fig. 1C).The inflammatory infiltrate contained macrophages and a few multinucleated giant cells; it involved the most posterior stroma and the area where Descemet's membrane would normally be present.Irregular hyphal fragments were seen in the section stained with methenamine silver (Fig. 1D) but not in a section stained with periodic acid-Schiff reagent.The hyphae were in the posterior stroma and within the adherent inflammatory infiltrate.The hyphae varied in width (4-6 μM), creating irregular contours.Septa were seen in only an occasional fragment.No fungi were identified in the lens, presumably because of its removal using a cryoprobe.
The patient experienced graft rejection fourteen months after the penetrating keratoplasty, which was successfully treated with aggressive steroid therapy.He underwent a wound revision four months post-op, and eight months post-op required a secondary posterior chamber intraocular lens placement.Eighteen months later, he experienced an inferior rhegmatogenous retinal detachment in his left eye, treated with a scleral buckle procedure, external retinal cryopexy, and drainage of subretinal fluid.Four years after discontinuing post-op antifungal therapy, his best corrected visual acuity in the left eye was stable at 20/40.

Discussion
Lasiodiplodia theobromae is a common fungus but a relatively rare cause of keratitis, with only 51 cases identified in our literature review (Table 1).Most of these cases were from the tropics or south Florida (Fig. 2, Table 2), which is reasonable since the fungus is most abundant C. Hamerski and A.D. Proia in these climates. 2Our case is unusual due to its occurrence in a more temperate climate and represents the first reported case in the United States outside of Florida.North Carolina has a sub-tropical climate, and Lasiodiplodia keratitis in such a climate seems exceptionally rare, with one case reported in Hong Kong. 9The patient's initial treatment with Maxitrol® ointment may have facilitated his developing Lasiodiplodia keratitis since corticosteroids 10 are contraindicated in cases of corneal foreign bodies 11,12 until fungal infection is excluded. 13ur patient's Lasiodiplodia theobromae remained undetected by culture and microscopy until four months after the corneal injury.L. theobromae keratitis in previously reported cases has not been occult on culture and microscopy, with many infections diagnosed during initial corneal scraping.Our patient did not have corneal scrapings, as there was no apparent superficial involvement by the infection.Instead,  23 1 Florida 1980 --Dutta 24 1 India 1981 --Slomovic 8 1 Florida 1985 Medical Enucleation (N.L.P.) Thomas 25 1 India 1991 P.K.P. "Graft clear."Gonawardena 26 1 Sri Lanka 1994 --Rosa 27 1 Florida 1994 --Dunlop 28 2 Bangladesh 1994 --Hagan 29 6 Ghana 1995 --Srinivasan 30 6 India 1997 --Borderie 6 1 Guyana 1997 Lensectomy Enucleation (N.L.P.) Gopinathan 31 7 India 2002 Donnio 7 1 Martinique 2006 Medical Evisceration (N.L.P.) Thew 32 2 Australia 2008 --Saha 19 1 India 2012 P.K.P. "Graft failed after 3 months."Samudio 33 1 Paraguay 2014 PKP -Lekhanont 5 2 Thailand 2015 Medical, PKP 20/200, counting fingers Li 9 1 Hong Kong 2016 PKP 6/30 da Rosa 17 1 Brazil 2018 PKP Hand movements Tangmonkongvoragul 34 6 Thailand 2021 PKP or patch graft (5/6) --No data available; LPlight perception; NLPno light perception; PKPpenetrating keratoplasty.pathogen identification was attempted using cultures of aqueous humor from anterior chamber taps.From the progression of slit-lamp findings in this case, we speculate that the inciting trauma seeded the deep corneal stroma with the fungus, which then spread posteriorly into the anterior chamber.A low fungal load likely caused the negative aqueous humor cultures until late in the case.Biopsy of the sub-endothelial plaque also noted no microbes on culture and microscopy, most likely due to the samples being inflammatory infiltrate devoid of organisms.][16] Pre-operative antimicrobial management (topical natamycin, amphotericin B, and flucytosine; oral ketoconazole and flucytosine; and subconjunctival miconazole) failed to control the infection in our patient.Oral itraconazole, along with topical amphotericin B and flucytosine, prevented postoperative recurrence of the infection.8][19] In vitro antifungal testing in two cases showed a sensitivity of L. theobromae to both amphotericin B and voriconazole, 17,19 and voriconazole has had varying degrees of success in four L. theobromae keratitis cases. 5,9,19However, voriconazole was not an option for our patient's treatment since it had not yet been approved for medical use.Amphotericin B has played a role in successful treatment in multiple cases, though never as monotherapy. 5,17,19fter the identification of L. theobromae in our case, miconazole and itraconazole were tried in succession due to their extended spectrum relative to other azole drugs.While miconazole was ineffective, itraconazole was part of the polytherapy that prevented recurrence postoperatively.It is unclear if the addition of itraconazole contributed to this success or if the decreased postoperative fungal load allowed clearance by another drug or the patient's immune system.In vitro testing in one article, published after our case, showed one L. theobromae strain to be resistant to itraconazole. 17Literature on the use of itraconazole against this organism is otherwise scarce.Indeed, the efficacy of any one drug against L. theobromae is challenging to discern in the literature.Further in-vitro sensitivity testing and documentation of more clinical cases are needed.

Fig. 1 .
Fig. 1. A. Clinical photograph three weeks prior to corneal transplantation.The central stroma has a dense white infiltrate in the deep layers of the cornea.B. Slitlamp photograph three weeks prior to corneal transplantation.The slit beam illuminates a dense white endothelial plaque with extension into the anterior chamber.Endothelial keratic precipitates are visible on the inferior cornea.C. Photomicrograph demonstrating granulomatous inflammation involving the posterior corneal stroma in a region where Descemet's membrane is absent (H&E, magnification bar = 100 μM).D. Photomicrograph showing irregular hyphae within the granulomatous inflammatory infiltrate (methenamine silver, magnification bar = 100 μM).

Fig. 2 .
Fig.2.Heat map showing reported cases of L. theobromae ocular infections in East Asia, Australia (A), and the east coast of the United States, including our patient, and Cuba (B).Reported cases elsewhere are not shown for legibility (see Table1).

Table 2
Geographic distribution of 51 previously reported cases of Lasiodiplodia theobromae keratitis.