to report the isolation of pestalotiopsis clavispora from the cornea of a patient with recurrent keratitis . a 73-year - old male gardener presented with conjunctival injection and an oval infiltrate with feathery margins in the temporal half of the cornea in the right eye . his ocular history in the right eye included cataract surgery , five episodes of herpes simplex keratitis , three glaucoma surgeries , and bullous keratopathy . light microscopy of corneal scrapings revealed a filamentous fungus , and fungal keratitis was diagnosed . the antifungal agents were discontinued 7 months later , and keratitis relapsed 4 days after the discontinuation . based on the results of antifungal susceptibility testing , treatment with topical and intravenous micafungin was initiated . molecular identification of the pathogen , and antifungal susceptibility testing , are useful in treating patients with fungal keratitis caused by a rare human pathogen . pestalotiopsis spp . , filamentous fungi , are well - known plant pathogens , and have been commonly isolated in subtropical and tropical regions.1 the genus pestalotiopsis was established in 1949 by steyaert , following a taxonomic amendment to the genus pestalotia . pestalotiopsis clavispora has been reported to cause a number of plant diseases , including diseases of blueberry and avocado plants.2,3 to our knowledge , this is the first case of human infection caused by p. clavispora . a 73-year - old male gardener presented to kurume university hospital in japan in april 2012 complaining of a foreign body sensation in his right eye after sweeping up leaves and twigs on a windy day . his ocular history in the right eye included cataract surgery ( 1990 ) , five episodes of herpes simplex keratitis ( 2006 , 2007 , 2008 [ two times ] , and 2010 ) , three glaucoma surgeries ( 2007 [ two times ] and 2008 ) , and bullous keratopathy ( 2011 ) . he had been treated with corticosteroids for years ( figure 1 ) . at initial presentation , his visual acuity was hand motion in the right eye and 20/250 in the left eye . intraocular pressure ( iop ) was not measured in the right eye , and was 6 mmhg in the left eye . slit - lamp examination of the right eye revealed conjunctival injection and an oval infiltrate with feathery margins in the temporal half of the cornea ( figure 2a ) . light microscopy of corneal scrapings taken from the right eye at initial presentation revealed uniformly thick septate hyphae ( figure 2b ) . the patient was diagnosed as having keratitis caused by a filamentous fungus , and was admitted to the hospital . a foreign body was found in the infiltrate when corneal debridement was performed ( figure 2c ) . topical corticosteroid treatment was discontinued , and topical treatment with voriconazole 1% hourly and pimaricin ( natamycin ) 1% ointment six times per day was initiated . the patient was discharged at 1 month , after the infiltrate had resolved . at the time of discharge , visual acuity in his right eye was hand motion and iop was 10 mmhg . treatment with topical voriconazole four times per day , and pimaricin two times per day , was continued . a search for the causative organism was initiated at the central clinical laboratory of kurume university hospital , but the organism was not identified . the isolate was then sent to the laboratory for clinical investigation at osaka university hospital , where it was examined morphologically . , based upon light microscopy with lactophenol cotton blue staining , which revealed conidia - bearing appendages ( figure 2d ) . a freshly isolated strain of pestalotiopsis spp . from the clinical specimen was subcultured on potato dextrose agar ( pda ) at 25c for 7 days . an inoculum suspension was prepared in roswell park memorial institute ( rpmi ) 1640 medium and adjusted to a final inoculum of 10,000 conidia . the minimum inhibitory concentrations ( mics ) for micafungin , amphotericin b , flucytosine , fluconazole , itraconazole , voriconazole , miconazole , and pimaricin were determined by the method of microdilution in rpmi 1640 broth , according to the protocol approved by the clinical and laboratory standards institute.4 the final concentrations of the drugs were 0.01516 g / ml for micafungin , 0.0316 g / ml for amphotericin b , miconazole , and pimaricin , 0.1264 g / ml for flucytosine and fluconazole , and 0.128 g / ml for itraconazole and voriconazole . , 100 l of adjusted inoculum were added , and the plates were incubated at 25c , without shaking . the isolate was sent to teikyo university institute of medical mycology for further morphological and molecular identification . a colony incubated on pda at 27c in the dark for one month revealed that the conidia were narrowly fusoid to fusoid - clavate , straight or somewhat curved , five - celled , with the upper cell conical to cylindrical , hyaline , fairly thin - walled , without visible cellular contents , and bearing two to four rather stout central apical appendages , 1020 m long and up to 1 m wide ( figure 2e ) . based on these morphological characteristics , the isolate was identified as pestalotiopsis spp.5 fungal dna was extracted from the isolate according to the rapid method described by makimura et al,6 and the internal transcribed spacer ( its ) region of the rrna gene was sequenced directly from the polymerase chain reaction products with the its1its4 universal primer pair.7 a basic local alignment search tool ( blast ) search revealed the sequences had 100% similarity only to p. clavispora ( ef119336 ) , registered in the ddbj / embl / genbank database , and less than 100% similarity to other species of pestalotiopsis . although the isolate demonstrated relatively high resistance to voriconazole ( mic : 2.0 g / ml ) and pimaricin ( mic : 2.0 g / ml ) through antifungal susceptibility testing ( table 1 ) , topical voriconazole and pimaricin continued to be used because the infiltrate decreased in size . treatment was discontinued 7 months after discharge , based upon the absence of conjunctival injection and corneal infiltrate . although fungus was not isolated from corneal scrapings at this time , treatment with topical micafungin ( mic : 0.03 g / ml ) 0.1% half - hourly , intravenous micafungin 50 mg daily , and corneal debridement weekly was commenced , based on the results of antifungal susceptibility testing . liver function and prothrombin time / international normalized ratio ( pt - inr ) were monitored periodically during systemic micafungin administration . intravenous micafungin was discontinued , and topical micafungin was tapered over 7 months , after resolution from half - hourly to three times per day . during this period , the conjunctival injection and the corneal infiltrate were not seen . visual acuity in his right eye was hand motion at the final visit , 7 months after resolution . no side effects related to topical voriconazole and micafungin were noted during the 15-month follow - up . a search for related studies was performed using the electronic database pubmed , until july 31 2013 , using the search term  pestalotiopsis clavispora  . no human or animal infections caused by this organism have been reported to this date . the patient was a gardener who presented with an eye infection after sweeping up leaves and twigs on a windy day . the right eye of this patient was predisposed to fungal infection , due to long - term use of corticosteroids , recurrent corneal surface disorders , and glaucoma ( figure 1 ) . rare human pathogens can cause keratitis in patients with a history of trauma or immunosuppression.8,9 antifungal susceptibility testing of our organism showed low susceptibility to voriconazole and pimaricin , but high susceptibility to micafungin ( table 1 ) . however , our initial treatment with topical voriconazole and pimaricin seemed to be effective in decreasing the infiltrate , because of the high concentrations that resulted from frequent administration of the antifungal agents . the keratitis might have been smoldering , because of lower doses of voriconazole and pimaricin . lowering the doses of voriconazole and pimaricin appears to have triggered the relapse , suggesting the treatment approach was inadequate , and that the initial medication should have been changed based on the results of the susceptibility testing . mochizuki et al reported that the corneal concentration of micafungin was 1.605.99 g / g , the aqueous concentration was 0.020.35 g / ml , and the vitreous concentration was 0.030.15 g / ml , when the dose of micafungin was 150300 mg / day.10 the mic for micafungin against the isolate from our patient was 0.03 g / ml . after the keratitis relapsed , our patient received treatment with topical micafungin 0.1% half - hourly , intravenous micafungin 50 mg daily , and corneal debridement weekly . although the dose of intravenous micafungin was much lower than the dose reported by mochizuki et al , we consider micafungin to have penetrated the cornea and the aqueous humor because of the combined use of micafungin . in addition , corneal debridement might have promoted the penetration of micafungin . in our case , based upon light microscopy with lactophenol cotton blue staining that revealed conidia bearing appendages , but the species was not yet identified ( figure 2d and e ) . using molecular biology techniques , , it is often challenging to identify an organism in human keratitis caused by rare fungal pathogens . dna sequencing was useful in identifying the pathogen in our case . in a strict sense , epitypification with living strains is necessary before clear results can be obtained.11 however , it is difficult to perform epitypification in clinical or biological laboratories . the isolated organism showed 100% similarity using the max ident score of the blast search . the results suggest that even a plant pathogen can cause human infection in immunocompromised hosts . antifungal susceptibility testing is beneficial for the treatment of fungal keratitis caused by rare fungal pathogens , and molecular biological techniques may be the only means to identify a rare fungus causing infection in humans .
purposeto report the isolation of pestalotiopsis clavispora from the cornea of a patient with recurrent keratitis.case reporta 73-year - old male gardener presented with conjunctival injection and an oval infiltrate with feathery margins in the temporal half of the cornea in the right eye . 
 his ocular history in the right eye included cataract surgery , five episodes of herpes simplex keratitis , three glaucoma surgeries , and bullous keratopathy . 
 he had been treated with corticosteroids for years . 
 light microscopy of corneal scrapings revealed a filamentous fungus , and fungal keratitis was diagnosed . 
 treatment with topical voriconazole and pimaricin ointment was commenced . 
 one month later , the infiltrate resolved . 
 the antifungal agents were discontinued 7 months later , and keratitis relapsed 4 days after the discontinuation . 
 the fungus was isolated and identified by molecular techniques as p. clavispora . based on the results of antifungal susceptibility testing , treatment with topical and intravenous micafungin was initiated . 
 the corneal infiltrate resolved 1 month after the relapse.conclusionmolecular identification of the pathogen , and antifungal susceptibility testing , are useful in treating patients with fungal keratitis caused by a rare human pathogen .