Diagnostic Microbiology and Infectious Disease 83 (2015) 49–52
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
The first case of endophthalmitis due to Rhinocladiella basitona in an immunocompetent patient☆ Mengyang Liu a,⁎, Xiaowen Xin b, Jing Li a, Shengjie Chen a a b
The Affiliated Hospital of Qingdao University, Qingdao, China Weihai Municipal Hospital, Weihai, China
a r t i c l e
i n f o
Article history: Received 14 February 2015 Received in revised form 30 April 2015 Accepted 23 May 2015 Available online 29 May 2015 Keywords: Fungal endophthalmitis Rhinocladiella basitona Penetrating keratoplasty
a b s t r a c t Rhinocladiella, a genus of black yeast–like fungi, is related to many infections in humans, including not only mild cutaneous lesions but also fatal brain infections. However, endophthalmitis caused by Rhinocladiella has never been reported by far. Herein, we present the first case of endophthalmitis due to Rhinocladiella basitona. The diagnosis was based on histopathology, mycology, and molecular identification. A 53-year-old female was struck by a piece of wood in her right eye. The wound in the central cornea became an ulcer and was aggravated continuously. Hyphae were found in the corneal scraping smear. Then endophthalmitis occurred and could not be controlled by the combined intravitreal antibiotic injections and vitrectomy. Finally, penetrating keratoplasty combined with retinal reattachment surgery was performed. Topical and systemic antifungal agents were administered for more than 1 month. The patient was cured, with improved visual acuity and clear corneal graft. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Fungal endophthalmitis (FE) is uncommon but may result in poor outcomes, including vision loss (Chhablani, 2011; Lingappan et al., 2012). It is usually divided into exogenous and endogenous types depending upon the mode of infection. Postocular operation (Chakrabarti et al., 2008) and trauma (Ramakrishnan et al., 2009) are the major etiologic factors of exogenous FE. For endogenous FE, however, the factors are various, such as abuse of intravenous drugs and extensive use of immunosuppressants and broad-spectrum antibiotics (Lingappan et al., 2012; Narendran et al., 2008; Zhang and Liu, 2010). The frequently encountered pathogens for endogenous FE, posttraumatic FE, and FE secondary to keratitis are Candida albicans, Aspergillus niger, and Fusarium solani, respectively (Essman et al., 1997; Sun et al., 2014; Wykoff et al., 2008; Zhang and Liu, 2010). Rhinocladiella species, mostly being Rhinocladiella aquaspersa and Rhinocladiella mackenziei, are usually seen in tropical regions. R. aquaspersa is often associated with skin infections (Badali et al., 2010a), while R. mackenziei causes brain infections in otherwise healthy individuals and leads to high mortality (Campbell and Al-Hedaithy, 1993; Didehdar et al., 2015). R. basitona, a member of the genus Rhinocladiella, was first identified as a human pathogenic fungus in 1998 (Suzuki et al., 1998). It initially belonged to Geniculosporium and was classified to Rhinocladiella by de Hoog et al. (2003). The clinical case caused by R. basitona is extremely rare. Only 2 cases of phaeohyphomycosis due to this pathogen have been reported (Cai et al., 2013; Suzuki et al., 1998). Herein, we present ☆ Conflicts of interest: None. ⁎ Corresponding author. Tel.: +86-532-82911511; fax: +86-532-82911999. E-mail address: [email protected] (M. Liu). http://dx.doi.org/10.1016/j.diagmicrobio.2015.05.014 0732-8893/© 2015 Elsevier Inc. All rights reserved.
a case of endophthalmitis related to Rhinocladiella basitona; describe its mycological characteristics; and evaluate the activity of triazole, amphotericin B, and natamycin against this microorganism in vitro. 2. Case report A 53-year-old female was struck by a piece of wood in her right eye and lost the vision in September 2008. The patient received corneal suture immediately and cataract extraction combined with intraocular lens implantation 15 days later at a local hospital. However, the wound in the central cornea became an ulcer and was aggravated continuously. Hyphae were found in the corneal scraping smear by microscopy. Eye drops and intravenous injection of fluconazole were given, but the infection could not be controlled. The patient was referred to our hospital on February 3, 2009. The visual acuity was finger counting/20 cm in the right eye and 20/20 in the left eye. Anteriorsegment examination showed conjunctival hyperemia and corneal edema with 2 mm × 3 mm of full-thickness infiltration in the corneal center. The iris and lens details were hazy with fibrin. Significant cytemediated inflammatory reactions were found in the anterior chamber. Lots of effusions were accrete in the ante-sac (Fig. 1A). Severe vitreous opacity and posterior vitreous detachment were observed by B-scan ultrasonography. No retinal detachment was detected. No hyphae were found by corneal scraping or confocal microscopy. On February 5, intravitreal antibiotic therapy was performed, and 0.1 g ceftazidime was injected into the vitreous cavity. Aqueous fluid and vitreous body were observed for microbiological examination. A large number of white blood cells were seen both in aqueous fluid and vitreous body, but no bacteria or fungi were found. Tobramycin eye drops were administered, and intravenous ciprofloxacin (15 mg/kg
50
M. Liu et al. / Diagnostic Microbiology and Infectious Disease 83 (2015) 49–52
Fig. 1. Clinical features and the examination results of the patient before treatment. A. Photograph of the right eye at first presentation to our hospital showing 2 mm × 3 mm of full-thickness infiltration in the central cornea and an irregular pupil. B. Confocal microscopic image showing hyphae in the deep layer of the cornea. C. Brown hyphae in the corneal scraping smear (10% KOH, original magnification ×400).
per day) was given for a week. At 1 week after the operation, the visual acuity was counting finger/30 cm. The samples did not show any growth of microorganisms up to 7 days. The patient had steadily improved vitreous clarity and decreased inflammation in the anterior chamber after treatments. However, the vitreous body became turbid again, and the infection became aggravated on February 26. The intravitreal antifungal therapy combined with vitrectomy was performed, and 1 mg voriconazole was injected into the vitreous cavity. The excised part was sent immediately for pathogenic examination. The sample smear and culture results were both negative. Confocal microscopic examination showed no hyphae. On March 9, the visual acuity was 20/500. The cornea was edematous with 3 mm × 4 mm of full-thickness infiltration in the center. The vitreous cavity was clear, and no retinal detachment was found. The patient complained the cloudiness in vision of the right eye on April 2. The cornea was edematous and cloudy with 4 mm × 5 mm of infiltration in the center. A round lesion was about 2 mm in diameter, within the substrate at the nasal side. The vitreous body was opaque. On April 6, hyphae were detected in the cornea by confocal microscopy (Fig. 1B), and brown hyphae were also found in the scraping of the cornea (Fig. 1C). The clinical specimen obtained from the scraping was incubated on chocolate agar (GC medium; Difco, Sparks, MD, USA) and 5% sheep blood agar (Oxoid, Basingstoke, Hampshire, England) at 37 °C for 72 hours and Sabouraud glucose agar (Oxoid) at 27 °C for 7 days. Eye drops of 0.5% fluconazole and oral itraconazole (200 mg per day) were administered. However, the infiltration at the nasal side of the cornea became larger 10 days later, indicating that the antimycotic treatment was not effective. Penetrating keratoplasty combined with retinal reattachment surgery was performed. The excised cornea was subjected for histopathological examination, and brown hyphae were found (Fig. 2C). After the operation, intravenous amphotericin B deoxycholate (0.6 mg/kg), oral itraconazole (200 mg), and 5% natamycin eye drops were administered daily. At 1 week after the operation, the visual acuity was 20/400, and the corneal allograft was transparent. The vitreous
body became clear, the fundus was clearer than before, and the retina was flat. Oral itraconazole and 5% natamycin eye drops were continued for 1 month. The signs of infection were alleviated, and the visual acuity was improved. After 1 year and 6 months, the visual acuity reached 20/100. Both the corneal allograft and the aqueous fluid were clear. 3. Mycology The excised corneal button was cut along the central line, a half for incubation on Sabouraud at 27 °C and another half for histopathological examination. The colony was 22 mm in diameter after 2 weeks, with olive-gray, smooth surface, which was black in the reverse side. Under the microscope, thick-walled, brown conidiophores were observed to arise at acute angles from creeping hyphae. The conidia were triangular, hyaline, and smooth, distributing in sympodial sequence. Conidiogenous cells were cylindrical with darkened scars. The lower conidia were shorter than the ultimate ones, with obvious scars (Fig. 2A and B). According to the morphological features observed in this isolate and the criteria put forward by De Hoog et al. (2000), the fungus was identified as R. basitona. Molecular analysis of the internal transcribed spacer rRNA gene was performed by PCR using primers ITS4 (5′-TCCTCCGCTTATTGATATGC3′) and ITS5 (5′-TCCGTAGGTGAACCTGCGG-3′). Amplification was carried out at 95 °C for 10 min, followed by 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The PCR products were purified and sequenced (Sinogene Scientific, Beijing, China). The sequence (GenBank ID KM036370, 615 bp) was analyzed with the basic local alignment search tool for nucleotide comparison on line, showing high homology with the fragment of R. basitona strain (GenBank EU041806), with 88% query cover and 100% identity. Histopathological observation confirmed the diagnosis of fungal endophthalmitis. Serially graded ethanol baths followed by xylene
Fig. 2. The culture and histopathological examination of the excised cornea. A. The smooth, expanding, olive-gray, and wool-like colony of R. basitona cultures on Sabouraud glucose agar after 14 days of incubation at 27 °C. B. Microculture of R. basitona. Thick-walled, septate hyphae arising at acute angles and generating cylindrical cells, with slightly darkened scars and triangular, hyaline, and smooth conidia (lactophenol cotton blue staining, original magnification ×400). C. Brown septate pigmented hyphae in the basilemma (H–E staining, original magnification ×400).
M. Liu et al. / Diagnostic Microbiology and Infectious Disease 83 (2015) 49–52
51
Table 1 Results of the antifungal agent susceptibility test. Antifungal agents
Amphotericin B
Fluconazole
Itraconazole
Ketoconazole
Natamycin
Voriconazole
MIC (μg/mL)
0.5
64
0.5
1
0.5
0.25
Note: Refer to CLSI document M38-A2.
were used to dehydrate the tissues before they were immersed in paraffin wax. The samples were embedded in paraffin molds, sectioned at 4-mm thickness, and mounted on glass slides. Then the hematoxylin– eosin stain was employed for microscopic examination and evaluation. Histopathologically, a mixture of acute and granulomatous inflammatory infiltrates was shown in the cornea. Pigmented fungal hyphae were observed within the deep corneal stroma (Fig. 2C). The in vitro antifungal susceptibilities of this strain were determined by broth microdilution according to the recommendations outlined in the CLSI (2008) document M38-A2. Amphotericin B (Sigma-Aldrich Chemie, Shanghai, China), fluconazole (Sigma-Aldrich), itraconazole (Sigma-Aldrich), and ketoconazole (Sigma-Aldrich); voriconazole (Pfizer Central Research, Sandwich, UK); and natamycin (North China Pharmaceutical Corporation, Shijiazhuang, Hebei, China) were used as regent-grade powders. The antifungal agents were dispensed into 96-well microdilution trays at a final concentration of 0.0313–16 mg/L (amphotericin B, itraconazole, voriconazole, and ketoconazole) or 0.125–64 mg/L (fluconazole and natamycin). Conidial suspensions in physiological saline containing Tween 40 (0.05%) were adjusted spectrophotometrically (530 nm) to optical densities in the range of 0.15–0.17, before diluted 1:50 in buffered RPMI 1640 medium to prepare final inoculum suspensions containing 0.5 × 104–4 × 10 4 CFU/mL, as verified by colony counts on Sabouraud glucose agar. Microtiter plates inoculated with R. basitona were incubated at 35 °C. After 96 h, the plates were examined visually, and the MIC of antifungal agents was determined. Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), and Paecilomyces variotii (ATCC 22319) were used for quality control (Table 1). 4. Discussion Endophthalmitis caused by dematiaceous fungi is rare (Alex et al., 2013; Ehlers et al., 2011; Revankar and Sutton, 2010; Sun et al., 2010). Its clinical features seem to be different from endophthalmitis caused by common pathogens like Fusarium, Aspergillus, and Candida. It is usually related to trauma, like other exogenous FE, but the traumatic agents are mostly decayed wood and plants. Dematiaceous infection tends to affect the deeper corneal stroma and has a longer incubation period and poorer prognosis than other traumatic FE. This kind of endophthalmitis may occur at a long period of time after fungal keratitis for the slow fungal growth in corneal tissue (Ho et al., 1991). Brown pigment makes pigmented plaques and can be seen clinically in fungal keratitis related to dematiaceous fungi (Berger et al., 1991). Since it is not easy to diagnose endophthalmitis caused by dematiaceous fungal pathogens, the treatment may be delayed, and the clinical outcomes are not favorable (Alex et al., 2013; Sun et al., 2010). In this case, R. basitona was inoculated deep into the cornea and caused corneal ulcer in the injured eye. Due to the resistance with fluconazole, the early medication was not effective, and the fungal hyphae inside the cornea could not be cleared up. The slowly growing fungi penetrated deep into the corneal stroma, resulting in endophthalmitis. When the patient came to our hospital for further treatment, because of the special growth pattern and pathopoiesis feature of the fungal pathogens, no hyphae were found, and misdiagnosis was made. Intravitreal antifungal therapy combined with vitrectomy (Narendran et al., 2008) is recommended for management of FE. The in vitro susceptibility test is important for selection of sensitive antifungal agents, but currently, there are no CLSI interpretative criteria or guidelines for R. basitona by microbroth dilution. In the previous reports (Badali et al.,
2010a,b), Rhinocladiella species were usually sensitive to itraconazole and voriconazole and resistant to amphotericin B sometimes but fluconazole always. In this study, R. basitona was sensitive to amphotericin B. Although amphotericin B is effective against a wide range of fungal pathogens (Wykoff et al., 2008), it was used intravenously only for 1 week in the patient for its potential toxicity (Hamill, 2013). Moreover, oral itraconazole was given for 1 month after penetrating keratoplasty. Penetrating keratoplasty is an effective approach to traumatic endophthalmitis by removing source of the infection. In a large series of fungal infections, 88% of superficial infections responded to medical therapy alone, versus 46% of cases with deeper infiltrates. Surgical treatment was required more often in patients who sought treatment late and had involvement of deep stroma (Garg et al., 2000). In our study, the patient received penetrating keratoplasty finally as R. basitona was inoculated deep into the cornea. In conclusion, dematiaceous fungal infection should be suspected when corneal ulcer recurs repeatedly or endophthalmitis occurs after plant trauma. A prolonged course of local and systemic antifungal treatment is important for prevention of disease recurrence. Early removal of the source of the infection is essential, and penetrating keratoplasty can be considered. Moreover, some patients in such situations may lose their confidence and give up treatment (Sun et al., 2010), so psychological counseling is very necessary.
References Alex D, Li D, Calderone R, Peters SM. Identification of Curvularia lunata by polymerase chain reaction in a case of fungal endophthalmitis. Med Mycol Case Rep 2013;2: 137–40. Badali H, Chander J, Gulati N, Attri A, Chopra R, Najafzadeh MJ, et al. Rhinocladiella aquaspersa, proven agent of verrucous skin infection and a novel type of chromoblastomycosis. Med Mycol 2010a;48:696–703. Badali H, de Hoog GS, Curfs-Breuker I, Meis JF. In vitro activities of antifungal drugs against Rhinocladiella mackenziei, an agent of fatal brain infection. J Antimicrob Chemother 2010b;65:175–7. Berger ST, Katsev DA, Mondino BJ, Pettit TH. Macroscopic pigmentation in a dematiaceous fungal keratitis. Cornea 1991;10:272–6. Cai Q, Lv GX, Jiang YQ, Mei H, Hu SQ, Xu HB, et al. The first case of Phaeohyphomycosis caused by Rhinocladiella basitona in an immunocompetent child in China. Mycopathologia 2013;176:101–5. Campbell CL, Al-Hedaithy SS. Phaeohyphomycosis of the brain caused by Ramichloridiwn mackenziei sp. nov. in Middle Eastern countries. J Med Vet Mycol 1993;31:325–32. Chakrabarti A, Shivaprakash MR, Singh R, Tarai B, George VK, Fomda BA, et al. Fungal endophthalmitis, fourteen years' experience from a center in India. Retina 2008;28: 1400–7. Chhablani J. Fungal endophthalmitis. Expert Rev Anti Infect Ther 2011;9:1191–201. Clinical Laboratory Standards Institute (CLSI). Reference method for broth dilution antifungal susceptibility testing of filamentous fungi-second edition: approved standard, M38-A2. Wayne, PA: CLSI; 2008. De Hoog GS, Guarro J, Gené J, Figueras MJ. Atlas of clinical fungi. 2nd ed. Utrecht: Centraalbureau voor Schimmelcultures; 2000. De Hoog GS, Vicente V, Caligiorne RB, Kantarcioglu S, Tintelnot K, Gerrits van den Ende AHG, et al. Species diversity and polymorphism in the Exophiala spinifera clade containing opportunistic black yeast-like fungi. J Clin Microbiol 2003;41:4767–78. Didehdar M, Gokanian A, Sofian M, Mohammadi R, Aslani N, Haghani I, et al. First fatal cerebral phaeohyphomycosis due to Rhinocladiella mackenziei in Iran, based on ITS rDNA. J Mycol Med 2015;25:81–6. Ehlers J, Chavala S, Woodward J, Postel Eric A. Delayed recalcitrant fungal endophthalmitis secondary to Curvularia. Can J Ophthalmol 2011;46:199–200. Essman TF, Flynn Jr HW, Smiddy WE, Brod RD, Murray TG, Davis JK, et al. Treatment outcomes in a 10-year study of endogenous fungal endophthalmitis. Ophthalmic Surg Lasers 1997;28:185–94. Garg P, Gopinathan U, Choudhary K, Rao GN. Keratomycosis: clinical and microbiologic experience with dematiaceous fungi. Ophthalmology 2000;107:574–80. Hamill RJ. Amphotericin B, formulations: a comparative review of efficacy and toxicity. Drugs 2013;9:919–34. Ho RH, Bernard PJ, McClellan KA. Phialophora mutabilis keratomycosis. Am J Ophthalmol 1991;112:728–9.
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Lingappan A, Wykoff CC, Albini TA, Miller D, Pathengay A, Davis JL, et al. Endogenous fungal endophthalmitis: causative organisms, management strategies, and visual acuity outcomes. Am J Ophthalmol 2012;153:162–6. Narendran N, Balasubramaniam B, Johnson E, Dick A, Mayer E. Five-year retrospective review of guideline-based management of fungal endophthalmitis. Acta Ophthalmol 2008;86:525–32. Ramakrishnan R, Bharathi MJ, Shivkumar C, Mittal S, Meenakshi R, Khadeer MA, et al. Microbiological profile of culture proven cases of exogenous and endogenous endophthalmitis: a 10-year retrospective study. Eye 2009;23:945–56. Revankar SG, Sutton DA. Melanized fungi in human disease. Clin Microbiol Rev 2010; 23:884–928.
Sun S, Zhao G, Yuan G, Chen Hao, Yu Bin. Endophthalmitis caused by P. verrucosa Medlar and Str. intermedius: a case report. Med Mycol 2010;48:1108–11. Sun S, Zhao G, Sun X, Wang Q, Yu B. Etiology and pathogens of fungal endophthalmitis. Zhonghua Yan Ke Za Zhi 2014;50:808–13. Suzuki Y, Udagawa S, Wakita H, Yamada N, Ichikawa H, Furukawa F, et al. Subcutaneous phaeohyphomycosis caused by Geniculosporium species: a new fungal pathogen. Br J Dermatol 1998;38:346–50. Wykoff CC, Flynn HW, Miller D, Scott IU, Alfonso EC. Exogenous fungal endophthalmitis: microbiology and clinical outcomes. Ophthalmology 2008;115:1501–7. (1507. e1501-1502). Zhang H, Liu Z. Endogenous endophthalmitis: a 10-year review of culture-positive cases in northern China. Ocul Immunol Inflamm 2010;18:133–8.
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
The first case of endophthalmitis due to Rhinocladiella basitona in an immunocompetent patient☆ Mengyang Liu a,⁎, Xiaowen Xin b, Jing Li a, Shengjie Chen a a b
The Affiliated Hospital of Qingdao University, Qingdao, China Weihai Municipal Hospital, Weihai, China
a r t i c l e
i n f o
Article history: Received 14 February 2015 Received in revised form 30 April 2015 Accepted 23 May 2015 Available online 29 May 2015 Keywords: Fungal endophthalmitis Rhinocladiella basitona Penetrating keratoplasty
a b s t r a c t Rhinocladiella, a genus of black yeast–like fungi, is related to many infections in humans, including not only mild cutaneous lesions but also fatal brain infections. However, endophthalmitis caused by Rhinocladiella has never been reported by far. Herein, we present the first case of endophthalmitis due to Rhinocladiella basitona. The diagnosis was based on histopathology, mycology, and molecular identification. A 53-year-old female was struck by a piece of wood in her right eye. The wound in the central cornea became an ulcer and was aggravated continuously. Hyphae were found in the corneal scraping smear. Then endophthalmitis occurred and could not be controlled by the combined intravitreal antibiotic injections and vitrectomy. Finally, penetrating keratoplasty combined with retinal reattachment surgery was performed. Topical and systemic antifungal agents were administered for more than 1 month. The patient was cured, with improved visual acuity and clear corneal graft. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Fungal endophthalmitis (FE) is uncommon but may result in poor outcomes, including vision loss (Chhablani, 2011; Lingappan et al., 2012). It is usually divided into exogenous and endogenous types depending upon the mode of infection. Postocular operation (Chakrabarti et al., 2008) and trauma (Ramakrishnan et al., 2009) are the major etiologic factors of exogenous FE. For endogenous FE, however, the factors are various, such as abuse of intravenous drugs and extensive use of immunosuppressants and broad-spectrum antibiotics (Lingappan et al., 2012; Narendran et al., 2008; Zhang and Liu, 2010). The frequently encountered pathogens for endogenous FE, posttraumatic FE, and FE secondary to keratitis are Candida albicans, Aspergillus niger, and Fusarium solani, respectively (Essman et al., 1997; Sun et al., 2014; Wykoff et al., 2008; Zhang and Liu, 2010). Rhinocladiella species, mostly being Rhinocladiella aquaspersa and Rhinocladiella mackenziei, are usually seen in tropical regions. R. aquaspersa is often associated with skin infections (Badali et al., 2010a), while R. mackenziei causes brain infections in otherwise healthy individuals and leads to high mortality (Campbell and Al-Hedaithy, 1993; Didehdar et al., 2015). R. basitona, a member of the genus Rhinocladiella, was first identified as a human pathogenic fungus in 1998 (Suzuki et al., 1998). It initially belonged to Geniculosporium and was classified to Rhinocladiella by de Hoog et al. (2003). The clinical case caused by R. basitona is extremely rare. Only 2 cases of phaeohyphomycosis due to this pathogen have been reported (Cai et al., 2013; Suzuki et al., 1998). Herein, we present ☆ Conflicts of interest: None. ⁎ Corresponding author. Tel.: +86-532-82911511; fax: +86-532-82911999. E-mail address: [email protected] (M. Liu). http://dx.doi.org/10.1016/j.diagmicrobio.2015.05.014 0732-8893/© 2015 Elsevier Inc. All rights reserved.
a case of endophthalmitis related to Rhinocladiella basitona; describe its mycological characteristics; and evaluate the activity of triazole, amphotericin B, and natamycin against this microorganism in vitro. 2. Case report A 53-year-old female was struck by a piece of wood in her right eye and lost the vision in September 2008. The patient received corneal suture immediately and cataract extraction combined with intraocular lens implantation 15 days later at a local hospital. However, the wound in the central cornea became an ulcer and was aggravated continuously. Hyphae were found in the corneal scraping smear by microscopy. Eye drops and intravenous injection of fluconazole were given, but the infection could not be controlled. The patient was referred to our hospital on February 3, 2009. The visual acuity was finger counting/20 cm in the right eye and 20/20 in the left eye. Anteriorsegment examination showed conjunctival hyperemia and corneal edema with 2 mm × 3 mm of full-thickness infiltration in the corneal center. The iris and lens details were hazy with fibrin. Significant cytemediated inflammatory reactions were found in the anterior chamber. Lots of effusions were accrete in the ante-sac (Fig. 1A). Severe vitreous opacity and posterior vitreous detachment were observed by B-scan ultrasonography. No retinal detachment was detected. No hyphae were found by corneal scraping or confocal microscopy. On February 5, intravitreal antibiotic therapy was performed, and 0.1 g ceftazidime was injected into the vitreous cavity. Aqueous fluid and vitreous body were observed for microbiological examination. A large number of white blood cells were seen both in aqueous fluid and vitreous body, but no bacteria or fungi were found. Tobramycin eye drops were administered, and intravenous ciprofloxacin (15 mg/kg
50
M. Liu et al. / Diagnostic Microbiology and Infectious Disease 83 (2015) 49–52
Fig. 1. Clinical features and the examination results of the patient before treatment. A. Photograph of the right eye at first presentation to our hospital showing 2 mm × 3 mm of full-thickness infiltration in the central cornea and an irregular pupil. B. Confocal microscopic image showing hyphae in the deep layer of the cornea. C. Brown hyphae in the corneal scraping smear (10% KOH, original magnification ×400).
per day) was given for a week. At 1 week after the operation, the visual acuity was counting finger/30 cm. The samples did not show any growth of microorganisms up to 7 days. The patient had steadily improved vitreous clarity and decreased inflammation in the anterior chamber after treatments. However, the vitreous body became turbid again, and the infection became aggravated on February 26. The intravitreal antifungal therapy combined with vitrectomy was performed, and 1 mg voriconazole was injected into the vitreous cavity. The excised part was sent immediately for pathogenic examination. The sample smear and culture results were both negative. Confocal microscopic examination showed no hyphae. On March 9, the visual acuity was 20/500. The cornea was edematous with 3 mm × 4 mm of full-thickness infiltration in the center. The vitreous cavity was clear, and no retinal detachment was found. The patient complained the cloudiness in vision of the right eye on April 2. The cornea was edematous and cloudy with 4 mm × 5 mm of infiltration in the center. A round lesion was about 2 mm in diameter, within the substrate at the nasal side. The vitreous body was opaque. On April 6, hyphae were detected in the cornea by confocal microscopy (Fig. 1B), and brown hyphae were also found in the scraping of the cornea (Fig. 1C). The clinical specimen obtained from the scraping was incubated on chocolate agar (GC medium; Difco, Sparks, MD, USA) and 5% sheep blood agar (Oxoid, Basingstoke, Hampshire, England) at 37 °C for 72 hours and Sabouraud glucose agar (Oxoid) at 27 °C for 7 days. Eye drops of 0.5% fluconazole and oral itraconazole (200 mg per day) were administered. However, the infiltration at the nasal side of the cornea became larger 10 days later, indicating that the antimycotic treatment was not effective. Penetrating keratoplasty combined with retinal reattachment surgery was performed. The excised cornea was subjected for histopathological examination, and brown hyphae were found (Fig. 2C). After the operation, intravenous amphotericin B deoxycholate (0.6 mg/kg), oral itraconazole (200 mg), and 5% natamycin eye drops were administered daily. At 1 week after the operation, the visual acuity was 20/400, and the corneal allograft was transparent. The vitreous
body became clear, the fundus was clearer than before, and the retina was flat. Oral itraconazole and 5% natamycin eye drops were continued for 1 month. The signs of infection were alleviated, and the visual acuity was improved. After 1 year and 6 months, the visual acuity reached 20/100. Both the corneal allograft and the aqueous fluid were clear. 3. Mycology The excised corneal button was cut along the central line, a half for incubation on Sabouraud at 27 °C and another half for histopathological examination. The colony was 22 mm in diameter after 2 weeks, with olive-gray, smooth surface, which was black in the reverse side. Under the microscope, thick-walled, brown conidiophores were observed to arise at acute angles from creeping hyphae. The conidia were triangular, hyaline, and smooth, distributing in sympodial sequence. Conidiogenous cells were cylindrical with darkened scars. The lower conidia were shorter than the ultimate ones, with obvious scars (Fig. 2A and B). According to the morphological features observed in this isolate and the criteria put forward by De Hoog et al. (2000), the fungus was identified as R. basitona. Molecular analysis of the internal transcribed spacer rRNA gene was performed by PCR using primers ITS4 (5′-TCCTCCGCTTATTGATATGC3′) and ITS5 (5′-TCCGTAGGTGAACCTGCGG-3′). Amplification was carried out at 95 °C for 10 min, followed by 30 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The PCR products were purified and sequenced (Sinogene Scientific, Beijing, China). The sequence (GenBank ID KM036370, 615 bp) was analyzed with the basic local alignment search tool for nucleotide comparison on line, showing high homology with the fragment of R. basitona strain (GenBank EU041806), with 88% query cover and 100% identity. Histopathological observation confirmed the diagnosis of fungal endophthalmitis. Serially graded ethanol baths followed by xylene
Fig. 2. The culture and histopathological examination of the excised cornea. A. The smooth, expanding, olive-gray, and wool-like colony of R. basitona cultures on Sabouraud glucose agar after 14 days of incubation at 27 °C. B. Microculture of R. basitona. Thick-walled, septate hyphae arising at acute angles and generating cylindrical cells, with slightly darkened scars and triangular, hyaline, and smooth conidia (lactophenol cotton blue staining, original magnification ×400). C. Brown septate pigmented hyphae in the basilemma (H–E staining, original magnification ×400).
M. Liu et al. / Diagnostic Microbiology and Infectious Disease 83 (2015) 49–52
51
Table 1 Results of the antifungal agent susceptibility test. Antifungal agents
Amphotericin B
Fluconazole
Itraconazole
Ketoconazole
Natamycin
Voriconazole
MIC (μg/mL)
0.5
64
0.5
1
0.5
0.25
Note: Refer to CLSI document M38-A2.
were used to dehydrate the tissues before they were immersed in paraffin wax. The samples were embedded in paraffin molds, sectioned at 4-mm thickness, and mounted on glass slides. Then the hematoxylin– eosin stain was employed for microscopic examination and evaluation. Histopathologically, a mixture of acute and granulomatous inflammatory infiltrates was shown in the cornea. Pigmented fungal hyphae were observed within the deep corneal stroma (Fig. 2C). The in vitro antifungal susceptibilities of this strain were determined by broth microdilution according to the recommendations outlined in the CLSI (2008) document M38-A2. Amphotericin B (Sigma-Aldrich Chemie, Shanghai, China), fluconazole (Sigma-Aldrich), itraconazole (Sigma-Aldrich), and ketoconazole (Sigma-Aldrich); voriconazole (Pfizer Central Research, Sandwich, UK); and natamycin (North China Pharmaceutical Corporation, Shijiazhuang, Hebei, China) were used as regent-grade powders. The antifungal agents were dispensed into 96-well microdilution trays at a final concentration of 0.0313–16 mg/L (amphotericin B, itraconazole, voriconazole, and ketoconazole) or 0.125–64 mg/L (fluconazole and natamycin). Conidial suspensions in physiological saline containing Tween 40 (0.05%) were adjusted spectrophotometrically (530 nm) to optical densities in the range of 0.15–0.17, before diluted 1:50 in buffered RPMI 1640 medium to prepare final inoculum suspensions containing 0.5 × 104–4 × 10 4 CFU/mL, as verified by colony counts on Sabouraud glucose agar. Microtiter plates inoculated with R. basitona were incubated at 35 °C. After 96 h, the plates were examined visually, and the MIC of antifungal agents was determined. Candida parapsilosis (ATCC 22019), Candida krusei (ATCC 6258), and Paecilomyces variotii (ATCC 22319) were used for quality control (Table 1). 4. Discussion Endophthalmitis caused by dematiaceous fungi is rare (Alex et al., 2013; Ehlers et al., 2011; Revankar and Sutton, 2010; Sun et al., 2010). Its clinical features seem to be different from endophthalmitis caused by common pathogens like Fusarium, Aspergillus, and Candida. It is usually related to trauma, like other exogenous FE, but the traumatic agents are mostly decayed wood and plants. Dematiaceous infection tends to affect the deeper corneal stroma and has a longer incubation period and poorer prognosis than other traumatic FE. This kind of endophthalmitis may occur at a long period of time after fungal keratitis for the slow fungal growth in corneal tissue (Ho et al., 1991). Brown pigment makes pigmented plaques and can be seen clinically in fungal keratitis related to dematiaceous fungi (Berger et al., 1991). Since it is not easy to diagnose endophthalmitis caused by dematiaceous fungal pathogens, the treatment may be delayed, and the clinical outcomes are not favorable (Alex et al., 2013; Sun et al., 2010). In this case, R. basitona was inoculated deep into the cornea and caused corneal ulcer in the injured eye. Due to the resistance with fluconazole, the early medication was not effective, and the fungal hyphae inside the cornea could not be cleared up. The slowly growing fungi penetrated deep into the corneal stroma, resulting in endophthalmitis. When the patient came to our hospital for further treatment, because of the special growth pattern and pathopoiesis feature of the fungal pathogens, no hyphae were found, and misdiagnosis was made. Intravitreal antifungal therapy combined with vitrectomy (Narendran et al., 2008) is recommended for management of FE. The in vitro susceptibility test is important for selection of sensitive antifungal agents, but currently, there are no CLSI interpretative criteria or guidelines for R. basitona by microbroth dilution. In the previous reports (Badali et al.,
2010a,b), Rhinocladiella species were usually sensitive to itraconazole and voriconazole and resistant to amphotericin B sometimes but fluconazole always. In this study, R. basitona was sensitive to amphotericin B. Although amphotericin B is effective against a wide range of fungal pathogens (Wykoff et al., 2008), it was used intravenously only for 1 week in the patient for its potential toxicity (Hamill, 2013). Moreover, oral itraconazole was given for 1 month after penetrating keratoplasty. Penetrating keratoplasty is an effective approach to traumatic endophthalmitis by removing source of the infection. In a large series of fungal infections, 88% of superficial infections responded to medical therapy alone, versus 46% of cases with deeper infiltrates. Surgical treatment was required more often in patients who sought treatment late and had involvement of deep stroma (Garg et al., 2000). In our study, the patient received penetrating keratoplasty finally as R. basitona was inoculated deep into the cornea. In conclusion, dematiaceous fungal infection should be suspected when corneal ulcer recurs repeatedly or endophthalmitis occurs after plant trauma. A prolonged course of local and systemic antifungal treatment is important for prevention of disease recurrence. Early removal of the source of the infection is essential, and penetrating keratoplasty can be considered. Moreover, some patients in such situations may lose their confidence and give up treatment (Sun et al., 2010), so psychological counseling is very necessary.
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