Int Ophthalmol DOI 10.1007/s10792-014-9961-5
ORIGINAL PAPER
Fusarium keratitis at a tertiary eye care centre in India Sujata Das • Savitri Sharma • Samir Mahapatra • Srikant K. Sahu
Received: 16 September 2013 / Accepted: 26 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The aim of the study was to report the clinical and microbiological profiles of Fusarium keratitis. In this single-centre, retrospective, noncomparative case series, 47 laboratory-confirmed cases of keratitis caused by Fusarium species treated at the L V Prasad Eye Institute, Bhubaneswar, India, between November 2006 and October 2009, were reviewed. The analysis included predisposing factors, clinical characteristics, microbiological findings, treatment and outcome. Forty-seven samples of 47 patients were included in the study. The mean age of the 47 patients was 46 ± 17 years. Twelve eyes had a history of injury. Corneal scraping could not be done in one of the cases due to large perforation. Fungal filaments were detected in corneal scraping in 41 cases, and in three cases microconidia were observed in microscopy. Fusarium solani was the most common species (44.7 %). All three cases where microconidia were present in smear were identified as F. solani in culture. The mean time to positive culture was 2.4 ± 1.5 days. Twenty-three patients underwent adjunctive surgical procedure. Visual acuity of \20/ 200 at presentation and final follow-up was noted in S. Das (&) S. Mahapatra S. K. Sahu Cornea and Anterior Segment Service, L V Prasad Eye Institute, Patia, Bhubaneswar 751 024, Odisha, India e-mail: [email protected]; [email protected] S. Sharma Ocular Microbiology Service, L V Prasad Eye Institute, Bhubaneswar 751 024, Odisha, India
80.9 and 51.4 % patients, respectively. One-half (23/ 47) of the patients had improvement in visual acuity. Fusarium keratitis may present after trauma without any satellite lesion, and the response to medical therapy is generally poor. Rapid diagnosis can be made by smear examination of corneal scrapings in a majority of the cases and confirmed by culture within 2–3 days. Presence of microconidia in smear examination may be suggestive of F. solani. Keywords Fusarium Fungus Microconidia Microbial keratitis
Introduction Microbial keratitis continues to be a leading cause of ocular morbidity and blindness worldwide [1]. Fungal infections of the cornea account for 20–50 % of all microbial keratitis cases in developing countries [1–4]. The epidemiological features of fungal keratitis vary across geographic regions and climatic conditions [5]. They may vary considerably across countries and within countries. It is essential to determine the local aetiology within a given region while planning a corneal ulcer management strategy. Several studies have investigated the epidemiology of corneal ulceration and causative microorganisms [1–4]. Fusarium and Aspergillus species are the most common fungi isolated from patients in tropical regions, while
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Candida species is the most common pathogen of fungal keratitis in the temperate zones. The prevalence of Fusarium keratitis has been reported to vary from 10 % [6] to 75 % [7] of fungal keratitis, thus making it an important genus affecting the cornea. Species of Fusarium are ubiquitous hyaline filamentous fungi, widely distributed in soil, and commonly associated with plant roots [8]. In humans, this organism has a predilection for infecting the cornea and is a well-known cause of disease among immunocompromised patients [9]. Several outbreaks of Fusarium keratitis have been reported among contact lens users in different parts of the world [10, 11]. This study aims to report the clinical and microbiological profiles of Fusarium keratitis seen at a tertiary eye care facility in India.
Materials and methods The medical and laboratory records of all cultureproven cases of Fusarium keratitis diagnosed at the L V Prasad Eye Institute, Bhubaneswar, India between November 2006 and October 2009 were reviewed retrospectively. All patients had undergone detailed clinical evaluation and slit lamp examination. Corneal scrapings were obtained from patients as a part of a standard protocol and subjected to direct microscopic examination (Gram and/or Giemsa, potassium hydroxide [10.0 %] with 0.1 % calcofluor white) and inoculation into various culture media, such as sheep blood agar (BA), chocolate agar (CA), Sabouraud dextrose agar (SDA), non-nutrient agar (NNA), thioglycollate broth and brain heart infusion (BHI) broth. SDA bottles were incubated at 26 ± 1 °C to enhance the growth of fungi, and the remaining plates were incubated at 36 ± 1 °C. While CA plates were incubated with 5 % carbon dioxide, BA, Thio and BHI broth were incubated aerobically, and NNA were incubated aerobically with an added live Escherichia coli suspension. All media were incubated for 2 weeks and were examined daily. A culture was considered significant and was reported if the smears demonstrated morphologically similar organisms, and/or if the same organism grew in more than one medium, and/or if there was growth on at least two streaks on blood agar. Significant fungi and bacteria were identified using standard techniques. Identification of Fusarium species was done with the aid of a pictorial guide [12]. Intensive
123
Table 1 Predisposing risk factors n (%) Ocular Trauma Vegetal matter Sand
12 (25.5 %) 9 1
Insect
1
Unknown
1
Chronic dacryocystitis
1 (2.1 %)
Corneal scar
1 (2.1 %)
Systemic Diabetes mellitus
5 (10.6 %)
topical medication with 5 % natamycin was started on the basis of the smear result. Topical medication was modified in some cases depending on the clinical response, culture report, and sensitivity of the isolated organism. The corneal tissues of those patients who underwent therapeutic keratoplasty were subjected to microbiological and histopathological examination. The retrospective data included patient demography (age, gender, and occupation), disease history (onset, course, predisposing factors), clinical appearance, microbiological finding, treatment and outcome.
Results Among the 887 corneal scrapings/corneal tissues/ anterior chamber exudates from clinically diagnosed microbial keratitis cases at our Institute, between November 2006 and October 2009, 195 cases (21.9 %) were culture positive for fungus. Fusarium species were recovered from 24.1 % (n = 47, i.e., 47 samples of 47 patients; corneal tissue: 4, corneal scraping: 41, anterior chamber exudates: 2) cases. It ranked second amongst fungal keratitis after Aspergillus species (35.4 %, n = 69). The mean age of 47 patients was 46 ± 17 years (range: 4–95, median: 45). The male to female ratio was 33:14. Nine (of 47 patients) were farmers in our case series. Predisposing factors The various ocular and systemic predisposing factors noted in our series are listed in Table 1. Twelve eyes (25.5 %) had history of trauma. Thirty-eight eyes
Int Ophthalmol Table 2 Visual acuity at initial and final examinations Range
Initial examination
Final examination
n
n
%
%
20/20–20/60
8
17.0
10
30.3
\20/60–20/200
1
2.1
7
21.2
38
80.9
16
48.5
\20/200 Total
47
media positivity rate for blood agar, chocolate agar, and SDA was 76.2 % (32/42), 78.6 % (33/42), and 76.7 % (33/43), respectively. The mean time to positive culture was 2.4 ± 1.5 days (range: 1–5, median: 2). Treatment
a
33
a
12 patients were lost to follow-up at final examination, and 2 patients had undergone evisceration
(80.9 %) had received some form of treatment prior to presentation. Four eyes (8.5 %) received topical corticosteroid before presentation. Twenty-eight patients (59.6 %) received topical antifungal agents. Clinical features All patients had symptoms of redness, tearing, photophobia, and decreased vision at presentation. The mean duration of symptom was 22 days (range: 3–300, median: 10). Visual acuity at the time of presentation is shown in Table 2. The size of the infiltrate in its largest diameter C6 mm was present in 42.6 % eyes (Fig. 1a–d). Eight eyes (17.0 %) had endothelial exudates. Hypopyon was present in 19 cases (40.4 %). None of the eyes had a satellite lesion.
All cases were started on antifungal agents. Topical natamycin was used as topical antifungal agent in all cases. Cases of mixed infection were treated with additional topical antibiotic drops. Ketoconazole was the most commonly used systemic antifungal agent. No response to medical therapy was defined by the following criteria (individual or in combination): increase in size and depth of stromal infiltrate, increase in anterior chamber exudate, progression of thinning, and perforation. Twenty-three patients (48.9 %) underwent adjunctive surgical procedure (application of tissue adhesive: n = 9; therapeutic penetrating keratoplasty: n = 15; anterior chamber wash: n = 4; evisceration: n = 2). The indications for therapeutic penetrating keratoplasty were as follows: large infiltrate (n = 8), no response to adequate medical therapy (n = 6), and large perforation (n = 1). The graft size was more than 9.0 mm in 60 % of the cases. Outcome
Microbiologic analysis Forty-one cases (89.1 %) were positive for fungus in direct microscopy of the corneal scraping. Results of microscopic examination are shown in Table-3. In three cases, microconidia were observed in direct smear examination (Fig. 1e, f). Fungal filaments were detected in 86.9 % cases both in KOH ? CFW and Gram stain. Chlamydospores were observed in 12 cases (26.1 %) (Fig. 1g, h). The different species of Fusarium identified in our series are shown in Table 3. The identification was based on colony characteristics and microscopic features. Fusarium solani was the most common (44.7 %) species. Six patients had associated bacterial infection (Staphylococcus spp., n = 3; Corynebacterium spp., n = 3; Pseudomonas sp., n = 1; Propionibacterium sp., n = 1). Two patients had more than one associated bacterial infection. All three cases where microconidia were present in smear were identified as Fusarium solani in culture. The culture
Outcome data were available for 35 cases, as 12 cases were lost to follow-up (Table 2). Of these, 15 (42.9 %) responded to medical treatment. Response to medical therapy was defined by the following criteria (individual or in combination): decrease in size and depth of stromal infiltrate, reduced anterior chamber exudate, scarring at the periphery of the infiltrate. The average duration of healing was 36.3 ± 28.8 days (median: 30). Of the 15 therapeutic keratoplasty, four grafts remained clear at the last follow-up. Visual acuity improved in 23 patients. There was no significant difference in the presenting features, clinical course, and final outcome between Fusarium solani and other Fusarium species (Table 4).
Discussion The species of Fusarium are widespread saprobic fungi that cause major diseases of plants, particularly
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Fig. 1 Slit lamp picture showing various clinical manifestations of Fusarium keratitis a corneal infiltrate with hyphae edge, b full-thickness infiltrate involving total cornea, c peripheral superficial corneal infiltrate, d large central corneal perforation. Direct microscopic examination of corneal scraping using
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different stains showing septate hyaline filaments e with microconidia in Gram stain, 10009; f with microconidia in Giemsa stain, 10009; g with chlamydospores in Gram stain, 10009; h with chlamydospores in potassium hydroxide and calcofluor white stain, 2009
Int Ophthalmol Table 3 Direct microscopic examination and culture result of corneal scraping Various species of Fusarium isolated F solani
F. roseum
TOTAL
F oxysporum
F moniliforme
F tricinctum
Fusarium sp.
Direct microscopic examination Fungal filaments
18
2
2
1
2
9
34 (73.9 %)
Fungal filaments ? Bacteria
1
–
1
1
–
4
7 (15.2 %)
Bacteria
–
–
–
–
–
2
2 (4.4 %)
No organism
2
–
–
–
–
1
3 (6.5 %)
– 21 (44.7 %)
– 2 (4.3 %)
– 3 (6.4 %)
1 3 (6.4 %)
– 2 (4.3 %)
– 16 (34.0 %)
1 47
Not donea Total a
In one case, corneal scraping could not be done due to large perforation; however, corneal tissue was culture positive for Fusarium sp
Table 4 Clinical profile and treatment outcome of patients with Fusarim solani and other Fusarium species Fusarim solani
Other Fusarium species
21
26
42.5 ± 17.1 (range: 13–75; median: 42) (34.7–50.3)
49.7 ± 26.3 (range: 4–95; median: 49) (39.1–60.3)
16.0 ± 17.1 (range 4–45; median: 12) (8.2–23.8)
26.3 ± 57.6 (range: 3–300; median: 10) (3.0–49.6)
3 (14.3 %)
9 (34.6 %)
No. (%)
11 (52.4 %)
9 (34.6 %)
Hypopyon No. (%)
7 (33.3 %)
12 (46.2 %)
3 (14.3 %)
5 (19.2 %)
11 (52.4 %)
11 (42.3 %)
18 (85.7 %) (63.6–96.9 %)
20 (76.9 %) (56.3–91.0 %)
7 (46.6 %) (21.2–73.4 %) [5 ? 1]
9 (50.0 %) (26.0–74.0 %) [7 ? 1]
Number of cases n Age (in years) Mean ± SD (95 % CI) Duration of symptom (in days) Mean ± SD (95 % CI) Trauma No. (%) Infiltrate size in its largest diameter C6 mm
Endothelial exudate No. (%) Surgical intervention No. (%) VA at presentation (\20/200) No. (%) [95 % CI] Final VA (\20/200) (excluding lost to follow-up patients) [Number of lost to follow-up patients ? Evisceration] No. (%) (95 % CI) SD standard deviation, CI confidence interval, VA visual acuity
major crop plants, and of humans. They have long been reported as important pathogens in eye infections, especially keratitis [2, 5, 7, 13]. They have also
been reported as the principal causes of fungal keratitis in many countries [2, 5, 7, 13]. They typically exhibit relative resistance to most antifungal agents,
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thereby posing a therapeutic challenge to the ophthalmologists. Trauma, often trivial and frequently associated with vegetal material, is well documented in fungal infection [2, 13–15]. In our series, 25.5 % had trauma, mostly, due to vegetal matter, which is similar across fungal keratitis in general. One out of 47 patients had nasolacrimal duct obstruction. Although nasolacrimal duct obstruction has been associated with microbial keratitis due to Streptococcus pneumoniae [16], association of fungal keratitis has not been reported before. This might be a coincidental finding. The patient did not have associated bacterial infection. Fusarium species have been reported as the most frequent fungal pathogen in non-therapeutic soft contact lens users [17]. None of our patients was contact lens user. Topical steroids have been known to favour fungal growth on the cornea. Incidence of prior steroid use in cases of fungal keratitis varies from 1.17 [15] to 60 % [18]. Four out of 47 patients in this series were using topical corticosteroid at the time of presentation. In our series, males were more affected than the females. This might be due to occupational history of male individuals that were related to outdoor activities. A significant proportion (42.6 %) of patients had a stromal infiltrate of[6 mm in the largest dimension at presentation. This might be due to late presentation in most of our cases. Although endothelial exudates and hypopyon were present in 8 and 19 cases, respectively, none of them had a satellite lesion. The satellites lesions may have merged together and form an extensive corneal infiltrate. Satellite lesion, a pathognomonic feature of fungal keratitis, is reported to be present in 10 % of fungal keratitis cases. Pe´rezBalbuena et al. [13] have reported the presence of satellite lesion in 75.4 % cases. However, we are unable to attach much significance to this finding. The rate of positive smears in microscopy in our series using Gram stain or KOH ? CFW was 89.1 %. This efficacy is comparable to earlier studies on fungal keratitis in general [6, 14, 19]. Presence of chlamydospores with hyaline septate hyphae in corneal scrapings may indicate Fusarium species although this finding is not exclusive for Fusarium. Microconidia were observed in three of our cases in direct smear examination. Fusarium is known to form reproductive structures in tissue, a phenomenon known as adventitious sporulation. This phenomenon may allow a rapid presumptive diagnosis of Fusarium
123
infection, while awaiting results from the culture. In addition, adventitious sporulation could be a virulence factor for disseminated infection, particularly in immunocompromised patients [20]. It is hypothesised that adventitious sporulation following hyphal invasion of vascular structures by Fusarium species leads to sustained release of conidia into the blood stream, resulting in fungaemia and rapid dissemination of infection [21]. In our study, all three patients with microconidia were F. solani in culture. The clinical course and outcome in these three cases were not different from the other cases. This study documents poor response to medical treatment in Fusarium keratitis necessitating keratoplasty in 15 cases (31.9 %). It is comparable to earlier report of Fusarium keratitis [10]. There was no significant difference in the presenting features, clinical course, and final outcome between Fusarium solani and other Fusarium species.
Conclusion Trauma with vegetal matter is a major risk factor in Fusarium keratitis. In our series, none of the patients were contact lens user. The classical satellite lesion is not found in our series. Although easily diagnosed in corneal scrapings, especially in the presence of hyaline septate hyphae with chlamydospores with or without microconidia, and culture confirmation within 2–3 days, treatment of Fusarium keratitis remains a challenge to the ophthalmologist. Acknowledgments This research Hyderabad Eye Research Foundation.
was
supported
by
Conflict of interest Authors do not have any financial or conflicting interests to disclose.
References 1. Srinivasan M, Gonzales CA, George C, Cevallos V, Mascarenhas JM, Asokan B, Wilkins J, Smolin G, Whitcher JP (1997) Epidemiology and aetiological diagnosis of corneal ulceration in Madurai, South India. Br J Ophthalmol 81(11):965–971 2. Gopinathan U, Garg P, Fernandes M, Sharma S, Athmanathan S, Rao GN (2002) The epidemiological features and laboratory results of fungal keratitis: a 10-year review at a referral eye care center in South India. Cornea 21(6): 555–559
Int Ophthalmol 3. Dunlop AA, Wright ED, Howlader SA, Nazrul I, Husain R, McClellan K, Billson FA (1994) Suppurative corneal ulceration in Bangladesh. A study of 142 cases examining the microbiological diagnosis, clinical and epidemiological features of bacterial and fungal keratitis. Aust N Z J Ophthalmol 22(2):105–110 4. Hagan M, Wright E, Newman M, Dolin P, Johnson G (1995) Causes of suppurative keratitis in Ghana. Br J Ophthalmol 79(11):1024–1028 5. Leck AK, Thomas PA, Hagan M, Kaliamurthy J, Ackuaku E, John M, Newman MJ, Codjoe FS, Opintan JA, Kalavathy CM, Essuman V, Jesudasan CA, Johnson GJ (2002) Aetiology of suppurative corneal ulcers in Ghana and south India, and epidemiology of fungal keratitis. Br J Ophthalmol 86(11):1211–1215 6. Ritterband DC, Seedor JA, Shah MK, Koplin RS, McCormick SA (2006) Fungal keratitis at the New York Eye and Ear Infirmary. Cornea 25(3):264–267 7. Mselle J (1999) Fungal keratitis as an indicator of HIV infection in Africa. Trop Doct 29(3):133–135 8. Thomas PA (2003) Current perspectives on ophthalmic mycoses. Clin Microbiol Rev 16(4):730–797 9. Nelson PE, Dignani MC, Anaissie EJ (1994) Taxonomy, biology, and clinical aspects of Fusarium species. Clin Microbiol Rev 7(4):479–504 10. Gorscak JJ, Ayres BD, Bhagat N, Hammersmith KM, Rapuano CJ, Cohen EJ, Burday M, Mirani N, Jungkind D, Chu DS (2007) An outbreak of Fusarium keratitis associated with contact lens use in the northeastern United States. Cornea 26(10):1187–1194 11. Khor WB, Aung T, Saw SM, Wong TY, Tambyah PA, Tan AL, Beuerman R, Lim L, Chan WK, Heng WJ, Lim J, Loh RS, Lee SB, Tan DT (2006) An outbreak of Fusarium keratitis associated with contact lens wear in Singapore. JAMA 295(24):2867–2873
12. Toussoun TA, Fusarium Nelson PE (1976) A pictorial guide to the identification of Fusarium species according to the taxonomic system of Snyder and Hansen. Pennsylvania State University Press, University Park 13. Pe´rez-Balbuena AL, Vanzzini-Rosano V, Valade´z-Virgen Jde J, Campos-Mo¨ller X (2009) Fusarium keratitis in Mexico. Cornea 28(6):626–630 14. Garg P, Gopinathan U, Choudhary K, Rao GN (2000) Keratomycosis: clinical and microbiologic experience with dematiaceous fungi. Ophthalmology 107(3):574–580 15. Bharathi MJ, Ramakrishnan R, Vasu S, Meenakshi R, Palaniappan R (2003) Epidemiological characteristics and laboratory diagnosis of fungal keratitis. A three-year study. Indian J Ophthalmol 51(4):315–321 16. Parmar P, Salman A, Kalavathy CM, Jesudasan CA, Thomas PA (2003) Pneumococcal keratitis: a clinical profile. Clin Experiment Ophthalmol 31(1):44–47 17. Alfonso EC, Miller D, Cantu-Dibildox J, O’brien TP, Schein OD (2006) Fungal keratitis associated with nontherapeutic soft contact lenses. Am J Ophthalmol 142(1): 154–155 18. Bhartiya P, Daniell M, Constantinou M, Islam FM, Taylor HR (2007) Fungal keratitis in Melbourne. Clin Experiment Ophthalmol 35(2):124–130 19. Tanure MA, Cohen EJ, Sudesh S, Rapuano CJ, Laibson PR (2000) Spectrum of fungal keratitis at Wills Eye Hospital, Philadelphia Pennsylvania. Cornea 19(3):307–312 20. Nucci M, Anaissie E (2007) Fusarium infections in immunocompromised patients. Clin Microbiol Rev 20(4): 695–704 21. Upton A, Marr KA (2006) Emergence of opportunistic mould infections in the hematopoietic stem cell transplant patient. Curr Infect Dis Rep 8(6):434–441
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ORIGINAL PAPER
Fusarium keratitis at a tertiary eye care centre in India Sujata Das • Savitri Sharma • Samir Mahapatra • Srikant K. Sahu
Received: 16 September 2013 / Accepted: 26 May 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract The aim of the study was to report the clinical and microbiological profiles of Fusarium keratitis. In this single-centre, retrospective, noncomparative case series, 47 laboratory-confirmed cases of keratitis caused by Fusarium species treated at the L V Prasad Eye Institute, Bhubaneswar, India, between November 2006 and October 2009, were reviewed. The analysis included predisposing factors, clinical characteristics, microbiological findings, treatment and outcome. Forty-seven samples of 47 patients were included in the study. The mean age of the 47 patients was 46 ± 17 years. Twelve eyes had a history of injury. Corneal scraping could not be done in one of the cases due to large perforation. Fungal filaments were detected in corneal scraping in 41 cases, and in three cases microconidia were observed in microscopy. Fusarium solani was the most common species (44.7 %). All three cases where microconidia were present in smear were identified as F. solani in culture. The mean time to positive culture was 2.4 ± 1.5 days. Twenty-three patients underwent adjunctive surgical procedure. Visual acuity of \20/ 200 at presentation and final follow-up was noted in S. Das (&) S. Mahapatra S. K. Sahu Cornea and Anterior Segment Service, L V Prasad Eye Institute, Patia, Bhubaneswar 751 024, Odisha, India e-mail: [email protected]; [email protected] S. Sharma Ocular Microbiology Service, L V Prasad Eye Institute, Bhubaneswar 751 024, Odisha, India
80.9 and 51.4 % patients, respectively. One-half (23/ 47) of the patients had improvement in visual acuity. Fusarium keratitis may present after trauma without any satellite lesion, and the response to medical therapy is generally poor. Rapid diagnosis can be made by smear examination of corneal scrapings in a majority of the cases and confirmed by culture within 2–3 days. Presence of microconidia in smear examination may be suggestive of F. solani. Keywords Fusarium Fungus Microconidia Microbial keratitis
Introduction Microbial keratitis continues to be a leading cause of ocular morbidity and blindness worldwide [1]. Fungal infections of the cornea account for 20–50 % of all microbial keratitis cases in developing countries [1–4]. The epidemiological features of fungal keratitis vary across geographic regions and climatic conditions [5]. They may vary considerably across countries and within countries. It is essential to determine the local aetiology within a given region while planning a corneal ulcer management strategy. Several studies have investigated the epidemiology of corneal ulceration and causative microorganisms [1–4]. Fusarium and Aspergillus species are the most common fungi isolated from patients in tropical regions, while
123
Int Ophthalmol
Candida species is the most common pathogen of fungal keratitis in the temperate zones. The prevalence of Fusarium keratitis has been reported to vary from 10 % [6] to 75 % [7] of fungal keratitis, thus making it an important genus affecting the cornea. Species of Fusarium are ubiquitous hyaline filamentous fungi, widely distributed in soil, and commonly associated with plant roots [8]. In humans, this organism has a predilection for infecting the cornea and is a well-known cause of disease among immunocompromised patients [9]. Several outbreaks of Fusarium keratitis have been reported among contact lens users in different parts of the world [10, 11]. This study aims to report the clinical and microbiological profiles of Fusarium keratitis seen at a tertiary eye care facility in India.
Materials and methods The medical and laboratory records of all cultureproven cases of Fusarium keratitis diagnosed at the L V Prasad Eye Institute, Bhubaneswar, India between November 2006 and October 2009 were reviewed retrospectively. All patients had undergone detailed clinical evaluation and slit lamp examination. Corneal scrapings were obtained from patients as a part of a standard protocol and subjected to direct microscopic examination (Gram and/or Giemsa, potassium hydroxide [10.0 %] with 0.1 % calcofluor white) and inoculation into various culture media, such as sheep blood agar (BA), chocolate agar (CA), Sabouraud dextrose agar (SDA), non-nutrient agar (NNA), thioglycollate broth and brain heart infusion (BHI) broth. SDA bottles were incubated at 26 ± 1 °C to enhance the growth of fungi, and the remaining plates were incubated at 36 ± 1 °C. While CA plates were incubated with 5 % carbon dioxide, BA, Thio and BHI broth were incubated aerobically, and NNA were incubated aerobically with an added live Escherichia coli suspension. All media were incubated for 2 weeks and were examined daily. A culture was considered significant and was reported if the smears demonstrated morphologically similar organisms, and/or if the same organism grew in more than one medium, and/or if there was growth on at least two streaks on blood agar. Significant fungi and bacteria were identified using standard techniques. Identification of Fusarium species was done with the aid of a pictorial guide [12]. Intensive
123
Table 1 Predisposing risk factors n (%) Ocular Trauma Vegetal matter Sand
12 (25.5 %) 9 1
Insect
1
Unknown
1
Chronic dacryocystitis
1 (2.1 %)
Corneal scar
1 (2.1 %)
Systemic Diabetes mellitus
5 (10.6 %)
topical medication with 5 % natamycin was started on the basis of the smear result. Topical medication was modified in some cases depending on the clinical response, culture report, and sensitivity of the isolated organism. The corneal tissues of those patients who underwent therapeutic keratoplasty were subjected to microbiological and histopathological examination. The retrospective data included patient demography (age, gender, and occupation), disease history (onset, course, predisposing factors), clinical appearance, microbiological finding, treatment and outcome.
Results Among the 887 corneal scrapings/corneal tissues/ anterior chamber exudates from clinically diagnosed microbial keratitis cases at our Institute, between November 2006 and October 2009, 195 cases (21.9 %) were culture positive for fungus. Fusarium species were recovered from 24.1 % (n = 47, i.e., 47 samples of 47 patients; corneal tissue: 4, corneal scraping: 41, anterior chamber exudates: 2) cases. It ranked second amongst fungal keratitis after Aspergillus species (35.4 %, n = 69). The mean age of 47 patients was 46 ± 17 years (range: 4–95, median: 45). The male to female ratio was 33:14. Nine (of 47 patients) were farmers in our case series. Predisposing factors The various ocular and systemic predisposing factors noted in our series are listed in Table 1. Twelve eyes (25.5 %) had history of trauma. Thirty-eight eyes
Int Ophthalmol Table 2 Visual acuity at initial and final examinations Range
Initial examination
Final examination
n
n
%
%
20/20–20/60
8
17.0
10
30.3
\20/60–20/200
1
2.1
7
21.2
38
80.9
16
48.5
\20/200 Total
47
media positivity rate for blood agar, chocolate agar, and SDA was 76.2 % (32/42), 78.6 % (33/42), and 76.7 % (33/43), respectively. The mean time to positive culture was 2.4 ± 1.5 days (range: 1–5, median: 2). Treatment
a
33
a
12 patients were lost to follow-up at final examination, and 2 patients had undergone evisceration
(80.9 %) had received some form of treatment prior to presentation. Four eyes (8.5 %) received topical corticosteroid before presentation. Twenty-eight patients (59.6 %) received topical antifungal agents. Clinical features All patients had symptoms of redness, tearing, photophobia, and decreased vision at presentation. The mean duration of symptom was 22 days (range: 3–300, median: 10). Visual acuity at the time of presentation is shown in Table 2. The size of the infiltrate in its largest diameter C6 mm was present in 42.6 % eyes (Fig. 1a–d). Eight eyes (17.0 %) had endothelial exudates. Hypopyon was present in 19 cases (40.4 %). None of the eyes had a satellite lesion.
All cases were started on antifungal agents. Topical natamycin was used as topical antifungal agent in all cases. Cases of mixed infection were treated with additional topical antibiotic drops. Ketoconazole was the most commonly used systemic antifungal agent. No response to medical therapy was defined by the following criteria (individual or in combination): increase in size and depth of stromal infiltrate, increase in anterior chamber exudate, progression of thinning, and perforation. Twenty-three patients (48.9 %) underwent adjunctive surgical procedure (application of tissue adhesive: n = 9; therapeutic penetrating keratoplasty: n = 15; anterior chamber wash: n = 4; evisceration: n = 2). The indications for therapeutic penetrating keratoplasty were as follows: large infiltrate (n = 8), no response to adequate medical therapy (n = 6), and large perforation (n = 1). The graft size was more than 9.0 mm in 60 % of the cases. Outcome
Microbiologic analysis Forty-one cases (89.1 %) were positive for fungus in direct microscopy of the corneal scraping. Results of microscopic examination are shown in Table-3. In three cases, microconidia were observed in direct smear examination (Fig. 1e, f). Fungal filaments were detected in 86.9 % cases both in KOH ? CFW and Gram stain. Chlamydospores were observed in 12 cases (26.1 %) (Fig. 1g, h). The different species of Fusarium identified in our series are shown in Table 3. The identification was based on colony characteristics and microscopic features. Fusarium solani was the most common (44.7 %) species. Six patients had associated bacterial infection (Staphylococcus spp., n = 3; Corynebacterium spp., n = 3; Pseudomonas sp., n = 1; Propionibacterium sp., n = 1). Two patients had more than one associated bacterial infection. All three cases where microconidia were present in smear were identified as Fusarium solani in culture. The culture
Outcome data were available for 35 cases, as 12 cases were lost to follow-up (Table 2). Of these, 15 (42.9 %) responded to medical treatment. Response to medical therapy was defined by the following criteria (individual or in combination): decrease in size and depth of stromal infiltrate, reduced anterior chamber exudate, scarring at the periphery of the infiltrate. The average duration of healing was 36.3 ± 28.8 days (median: 30). Of the 15 therapeutic keratoplasty, four grafts remained clear at the last follow-up. Visual acuity improved in 23 patients. There was no significant difference in the presenting features, clinical course, and final outcome between Fusarium solani and other Fusarium species (Table 4).
Discussion The species of Fusarium are widespread saprobic fungi that cause major diseases of plants, particularly
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Fig. 1 Slit lamp picture showing various clinical manifestations of Fusarium keratitis a corneal infiltrate with hyphae edge, b full-thickness infiltrate involving total cornea, c peripheral superficial corneal infiltrate, d large central corneal perforation. Direct microscopic examination of corneal scraping using
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different stains showing septate hyaline filaments e with microconidia in Gram stain, 10009; f with microconidia in Giemsa stain, 10009; g with chlamydospores in Gram stain, 10009; h with chlamydospores in potassium hydroxide and calcofluor white stain, 2009
Int Ophthalmol Table 3 Direct microscopic examination and culture result of corneal scraping Various species of Fusarium isolated F solani
F. roseum
TOTAL
F oxysporum
F moniliforme
F tricinctum
Fusarium sp.
Direct microscopic examination Fungal filaments
18
2
2
1
2
9
34 (73.9 %)
Fungal filaments ? Bacteria
1
–
1
1
–
4
7 (15.2 %)
Bacteria
–
–
–
–
–
2
2 (4.4 %)
No organism
2
–
–
–
–
1
3 (6.5 %)
– 21 (44.7 %)
– 2 (4.3 %)
– 3 (6.4 %)
1 3 (6.4 %)
– 2 (4.3 %)
– 16 (34.0 %)
1 47
Not donea Total a
In one case, corneal scraping could not be done due to large perforation; however, corneal tissue was culture positive for Fusarium sp
Table 4 Clinical profile and treatment outcome of patients with Fusarim solani and other Fusarium species Fusarim solani
Other Fusarium species
21
26
42.5 ± 17.1 (range: 13–75; median: 42) (34.7–50.3)
49.7 ± 26.3 (range: 4–95; median: 49) (39.1–60.3)
16.0 ± 17.1 (range 4–45; median: 12) (8.2–23.8)
26.3 ± 57.6 (range: 3–300; median: 10) (3.0–49.6)
3 (14.3 %)
9 (34.6 %)
No. (%)
11 (52.4 %)
9 (34.6 %)
Hypopyon No. (%)
7 (33.3 %)
12 (46.2 %)
3 (14.3 %)
5 (19.2 %)
11 (52.4 %)
11 (42.3 %)
18 (85.7 %) (63.6–96.9 %)
20 (76.9 %) (56.3–91.0 %)
7 (46.6 %) (21.2–73.4 %) [5 ? 1]
9 (50.0 %) (26.0–74.0 %) [7 ? 1]
Number of cases n Age (in years) Mean ± SD (95 % CI) Duration of symptom (in days) Mean ± SD (95 % CI) Trauma No. (%) Infiltrate size in its largest diameter C6 mm
Endothelial exudate No. (%) Surgical intervention No. (%) VA at presentation (\20/200) No. (%) [95 % CI] Final VA (\20/200) (excluding lost to follow-up patients) [Number of lost to follow-up patients ? Evisceration] No. (%) (95 % CI) SD standard deviation, CI confidence interval, VA visual acuity
major crop plants, and of humans. They have long been reported as important pathogens in eye infections, especially keratitis [2, 5, 7, 13]. They have also
been reported as the principal causes of fungal keratitis in many countries [2, 5, 7, 13]. They typically exhibit relative resistance to most antifungal agents,
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thereby posing a therapeutic challenge to the ophthalmologists. Trauma, often trivial and frequently associated with vegetal material, is well documented in fungal infection [2, 13–15]. In our series, 25.5 % had trauma, mostly, due to vegetal matter, which is similar across fungal keratitis in general. One out of 47 patients had nasolacrimal duct obstruction. Although nasolacrimal duct obstruction has been associated with microbial keratitis due to Streptococcus pneumoniae [16], association of fungal keratitis has not been reported before. This might be a coincidental finding. The patient did not have associated bacterial infection. Fusarium species have been reported as the most frequent fungal pathogen in non-therapeutic soft contact lens users [17]. None of our patients was contact lens user. Topical steroids have been known to favour fungal growth on the cornea. Incidence of prior steroid use in cases of fungal keratitis varies from 1.17 [15] to 60 % [18]. Four out of 47 patients in this series were using topical corticosteroid at the time of presentation. In our series, males were more affected than the females. This might be due to occupational history of male individuals that were related to outdoor activities. A significant proportion (42.6 %) of patients had a stromal infiltrate of[6 mm in the largest dimension at presentation. This might be due to late presentation in most of our cases. Although endothelial exudates and hypopyon were present in 8 and 19 cases, respectively, none of them had a satellite lesion. The satellites lesions may have merged together and form an extensive corneal infiltrate. Satellite lesion, a pathognomonic feature of fungal keratitis, is reported to be present in 10 % of fungal keratitis cases. Pe´rezBalbuena et al. [13] have reported the presence of satellite lesion in 75.4 % cases. However, we are unable to attach much significance to this finding. The rate of positive smears in microscopy in our series using Gram stain or KOH ? CFW was 89.1 %. This efficacy is comparable to earlier studies on fungal keratitis in general [6, 14, 19]. Presence of chlamydospores with hyaline septate hyphae in corneal scrapings may indicate Fusarium species although this finding is not exclusive for Fusarium. Microconidia were observed in three of our cases in direct smear examination. Fusarium is known to form reproductive structures in tissue, a phenomenon known as adventitious sporulation. This phenomenon may allow a rapid presumptive diagnosis of Fusarium
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infection, while awaiting results from the culture. In addition, adventitious sporulation could be a virulence factor for disseminated infection, particularly in immunocompromised patients [20]. It is hypothesised that adventitious sporulation following hyphal invasion of vascular structures by Fusarium species leads to sustained release of conidia into the blood stream, resulting in fungaemia and rapid dissemination of infection [21]. In our study, all three patients with microconidia were F. solani in culture. The clinical course and outcome in these three cases were not different from the other cases. This study documents poor response to medical treatment in Fusarium keratitis necessitating keratoplasty in 15 cases (31.9 %). It is comparable to earlier report of Fusarium keratitis [10]. There was no significant difference in the presenting features, clinical course, and final outcome between Fusarium solani and other Fusarium species.
Conclusion Trauma with vegetal matter is a major risk factor in Fusarium keratitis. In our series, none of the patients were contact lens user. The classical satellite lesion is not found in our series. Although easily diagnosed in corneal scrapings, especially in the presence of hyaline septate hyphae with chlamydospores with or without microconidia, and culture confirmation within 2–3 days, treatment of Fusarium keratitis remains a challenge to the ophthalmologist. Acknowledgments This research Hyderabad Eye Research Foundation.
was
supported
by
Conflict of interest Authors do not have any financial or conflicting interests to disclose.
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