Mycotic Keratitis in Eastern India: Report of a Decade

doi:10.21203/rs.3.rs-1292963/v1

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Mycotic Keratitis in Eastern India: Report of a Decade

https://doi.org/10.21203/rs.3.rs-1292963/v1

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Purpose

This study aimed to identify the spectrum of the causative organisms of fungal keratitis and to estimate the disease burden in Eastern India for last 10 years.

Method

Patients with microbial keratitis attended the cornea service of the tertiary care hospital in West Bengal were screened for Mycotic Keratitis by 10%KOH and culture in Dextrose Sabouraud’s Agar from 2010 till 2019. Data were collected from electronic data set, Outpatient and Inpatient department tickets and analysed for epidemiological features in retrospective fashion

Results

Within 3307 clinically suspected fungal keratitis cases, 1287 (38.92 %) cases were direct microscopy confirmed. Out of 1287 microscopic positive cases 811 (63.01%) were culture positive. In culture positive cases highest was Aspergillus (n = 371, 45.75%) followed by Fusarium (n = 171, 21.09%), Candida (n = 127, 15.86%), and Curvularia (n = 80, 9.86%). Male predominance (62.15%) reported. Peak of infection noted in post-monsoon season. Prominent geographical variation found in this study. Trauma with vegetative matter found to be most common cause in case of Aspergillus (P = 0.009). Candida infection was significantly seen in immuno-compromised patient (P = 0.002). Curvularia found to have highest time to heal (40.83 ± 19.59 days). In all 404 (49.81%) required therapeutic keratoplasty.

Conclusion

Fungal keratitis is a very common cause of corneal blindness in Eastern India, in which Aspergillus is the most common organism and trauma by vegetative matter is the most common risk factor. The spectrum of the causative organisms is very broad due to diverse climatic condition of West Bengal.

Trial registration number and date: CTRI/2020/12/029767, REGISTERED ON 14/12/2020, CLINICAL TRIALS REGISTRY -INDIA.

Fungus

keratitis

vegetative

trauma

aspergillus

immunocompromised

Corneal disease is second only to cataracts as the most common cause of blindness worldwide, resulting in over 1.5 million recent cases of vision loss annually [1]. Ophthalmic mycosis is emerging as a major cause of vision loss and morbidity and can be life-threatening [2,3]. Fungal keratitis is one of the major causes of ophthalmic mycosis, accounting for over 50% of proven ophthalmic mycoses in some countries [4,5]. The epidemiology differs at various geographical locations in India. In South India, fungi cause about 44% of corneal ulcers [6]. In North India, about 31% of corneal ulcers were because of fungus. Fusarium species are predominant in South India, whereas Aspergillus species are predominant in North India [7].

Treatment of fungal keratitis is more difficult than that of bacterial ulcers, and resulting visual impairment is, on average, more severe. The visual outcome is also poor because of delayed presentation. In this study, we aimed to evaluate the trend and epidemiological characteristics, laboratory findings and treatment outcomes among the fungal corneal ulcer patients who have come to the Cornea services of Regional Institute of Ophthalmology, Kolkata for a decade.

This study was conducted in Cornea service of a Tertiary Care Hospital in Eastern India after getting approval from Institutional the research and ethics committee following the Declaration of Helsinki. This study is registered under Clinical Trials Registry – India with CTRI number CTRI/2020/12/029767.

It is a retrospective institution base epidemiological study, where patients of clinically suspected fungal keratitis patients were tested to identify microbiologically to confirm fungal keratitis over the period of 2010 through 2019. Complete inclusion and exclusion criteria are listed below.

Inclusion Criteria:

Presence of an active corneal ulcer at presentation.
Evidence of filamentous fungus on examination of KOH mount (confirmed by culture subsequently).

Exclusion Criteria:

Overlying epithelial defect < 0.5 mm at its greatest width at presentation.
Impending perforation.
Evidence of Bacterial or Acanthamoebal infection or Herpetic keratitis by history or examination.
Corneal scar not easily distinguishable from current ulcer.
Previous penetrating keratoplasty in the affected eye.
Pregnancy (by history or urine test) or breast-feeding (by history).
Acuity worse than 6/60 (20/200) in the fellow eye.
Known allergy to study medications (antifungal or preservative).
No light perception in the affected eye.

Final case selection:

Only KOH positive cases which yielded positive fungal culture were included in the study.

All other cases which showed negative result in any of the two tests (KOH mount & fungal culture) are excluded from the study.

All the cases which showed evidence of mixed infection are also excluded from the study.

Intervention:

All patients with a corneal ulcer presenting to Regional Institute of ophthalmology, Kolkata India, had corneal scrapings using a 15-number surgical blade, corneal button, corneal tissue and corneal exudates were tested for Gram stain and potassium hydroxide wet mount and had cultures plated on blood, chocolate, and potato dextrose agar. For fungal corneal ulcer, direct microscopy with potassium hydroxide (10% KOH) was done. Samples were inoculated in two Sabouraud’s dextrose agar plate with chloramphenicol (SDC) in the form of ‘C’ shape. It was then incubated 25^oC and 37^oC respectively and reviewed daily till six weeks for any growth. If any fungal growth noted in ‘C’, was subcultured and identification was done by phenotypic, macroscopic, and microscopic features like texture, pigmentation, arrangement of mycelium and conidium types by lactophenol cotton blue mount. The culture was tagged as positive, where the growth of the same fungus noted in more than one solid media.

All patients were treated with the antifungal medications, as needed. Therapeutic penetrating keratoplasty had also been done in non-responsive cases.

All the patients were followed up till the healing of the corneal ulcer or three-month duration, whichever is earlier. The date of first examination was taken into consideration while calculating time required for the ulcer to heal. Follow up was done based on visual acuity, symptomatic improvement, size of ulcer, hypopyon, central opacification, and complications.

All the patients were closely followed up on a weekly basis at the outpatient department. Patients who missed two or more consecutive follow-up visit were excluded from the study. Also, the patients who showed evidence of noncompliance to the treatment protocol were excluded from the study.

Epidemiological data and clinical data were recorded from the history and OPD tickets findings which were recorded at first and subsequent visit of the patient.

A total 3307 patients of corneal ulcers were registered in our OPD and Cornea clinic from 2010 to 2019. Amongst them, 1287 (38.92%) diagnosed as mycotic keratitis based on clinical findings and positive microscopic findings in 10% KOH mount. 811 (63.01%) patients were culture positive, rest 476 (36.98%) were only smear positive [Table 1]. It is found that 504 (62.15%) were male and 307 (37.85%) were female. The mean age of the population is 49.81±20.72 (range 4 – 74). Patients >40 years old are predominant in this study.

Table 1

Incidence of Mycotic Keratitis from 2010 - 2019
Year	Total number of corneal ulcers (no)	10% KOH mount positive (no)	10% KOH mount positive (%)	SDC agar Positive (no)	SDC agar Positive (%)
2010	313	122	38.98%	71	58.20%
2011	408	145	35.54%	87	60.00%
2012	375	133	35.47%	79	59.40%
2013	329	157	47.72%	95	60.51%
2014	280	104	37.14%	63	60.58%
2015	296	99	33.45%	71	71.72%
2016	322	142	44.10%	92	64.79%
2017	220	95	43.18%	68	71.58%
2018	386	169	43.78%	101	59.76%
2019	378	121	32.01%	84	69.42%
Total	3307	1287	38.92%	811	63.01%

Varieties of agents of 32 genera (28 filamentous, 3 yeast, 1 dimorphic) were isolated. Aspergillus species was highest in number (n = 371, 45.75%), followed by Fusarium (n = 171, 21.09%) and Candida (n = 127, 15.77%) and Curvularia (n = 80, 9.86%) [Table 2]. Rest 62 samples included several filamentous, yeast (except Candida), and dimorphic fungi. Some very rare fiamentous fungi, like Acremonium, Beauvaria, Cladosporium, Scopulariopsis, Trichophyton, Rhizopus are found, in Yeast, except Candida there were Saccharomyces and Trichosporon, and in Dimorphic Fungi Sporothrix was found. [Table 3].

Table 2

Trend of causative organism causing mycotic keratitis during 2010–2019
	2010	2011	2012	2013	2014	2015	2016	2017	2018	2019	Total	%
Aspergillus	31	40	28	47	25	36	41	30	52	41	371	45.75%
Fusarium	16	21	18	24	11	16	23	10	18	14	171	21.09%
Candida	12	16	14	10	11	11	17	8	15	13	127	15.66%
Curvularia	6	4	10	9	8	5	7	12	10	9	80	9.86%
Other Fungi	6	6	9	5	8	3	4	8	6	7	62	7.64%
Total	71	87	79	95	63	71	92	68	101	84	811	100.00%

Table 3

Spectrum of Organisms isolated during the study period
Name of Organism	No of sample Total = 811	%
Aspergillus	371	45.75%
Fusarium	171	21.09%
Acremonium	6	0.74%
Beauvaria	1	0.12%
Cladosporium	1	0.12%
Cylindrocarpon	1	0.12%
Epidermophyton	2	0.25%
Fusidium	3	0.37%
Microsporum	1	0.12%
Nigrospora	1	0.12%
Penicillium	9	1.11%
Paecilomyces	2	0.25%
Scedosporium	4	0.49%
Scopulariopsis	1	0.12%
Trychophyton	5	0.62%
Sarcinomyces	1	0.12%
Curvularia	80	9.86%
Alternaria	16	1.97%
Mucor	2	0.25%
Rhizopus	1	0.12%
Candida	127	15.66%
Saccharomyces	3	0.37%
Trichosporon	1	0.12%
Sporothrix	1	0.12%

There is a prominent evidence of seasonal variation in case of incidence of fungal corneal ulcer, it is noted that gradual increase in number of cases from March and peak is noted around August to October, which is post-monsoon or paddy harvesting period in Eastern India [Fig. 1]. Aspergillus follows the same trend, whereas Fusarium, Candida and Curvularia does not follow any pattern. The incidence of fungal keratitis also follows a particular geographical pattern, patients with Aspergillus predominantly came from Burdwan, Birbhum and Murshidabad districts, Fusarium also follows the same trend it also involved North Bengal districts, Curvularia is more predominant in districts Purulia, Bankura, Midnapore and South 24 Parganas region, Candida does not follow any trend, but mainly seen in North Bengal, Sub Himalayan region of West Bengal. One dimorphic fungi case came from tea garden of North Bengal, and uncommon molds are mostly presented from North Bengal districts.

Various risk factors are noted for causing of fungal corneal ulcer like trauma with vegetative matter, unknown foreign body, spontaneous appearance, blunt trauma, exposure to mud, cow horn/ tail injury, bird beak injury, fish injury, insect falling, iron particle, prior steroid use, immunocompromise. While comparing the Four major groups of fungal keratitis it is noted that trauma with vegetative matter is statistically significant in case of Aspergillus with comparison to others (P = 0.009, ONE WAY ANOVA) whereas being immunocompromise is a statistically significant risk factors for Candida with comparison to others (P = 0.002, ONE WAY ANOVA) [Table 4].

Table 4

Details of risk factors among four major groups of patients with fungal keratitis
Risk Factors	Aspergillus (n = 371)		Fusarium (n = 171)		Candida (n = 127)		Curvularia (n = 80)
Trauma with vegetative matter	139	37.46%^*	55	32.16%	14	11.02%	10	12.50%
Unknown foreign body	55	14.82%	52	30.41%	17	13.39%	11	13.75%
Spontaneous appearance	23	6.19%	20	11.70%	12	9.45%	3	3.75%
Blunt trauma	39	10.51%	8	4.68%	12	9.45%	2	2.50%
Exposure to mud	20	5.39%	15	8.77%	2	1.57%	25	31.25%
Cow horn/ tail injury	24	6.47%	1	0.58%	8	6.29%	8	10.00%
Bird beak injury	9	2.43%	0	0.00%	6	4.72%	8	10.00%
Insect falling	31	8.35%	13	7.60%	3	2.36%	6	7.50%
Iron particle	25	6.74%	3	1.75%	1	0.79%	7	8.75%
Immunocompromise	1	0.27%	0	0.00%	35	27.55%^**	0	0.00%
Prior Steroid use	5	1.35%	4	2.34%	17	13.39%	0	0.00%
* Statistically significant in case of Aspergillus with comparison to others (P = 0.009) ** Statistically higher on comparing Candida with others (P = 0.002)

All the different types of fungal ulcers had stromal infiltrates >1.5 mm² with associated signs of inflammation. The duration of the illness is highest in case of Curvularia than the others (P = 0.046, ONE WAY ANOVA). 349 (43.03%) patients out of 811 of culture positive fungal ulcers were recovered by corneal scar formation, only with medical treatment. 404 (49.81%) required therapeutic keratoplasty on the course of treatment and 58 (7.15%) culture positive patients required immediate therapeutic keratoplasty at present. Within 462 (56.96%) patients who have required therapeutic keratoplasty, 186 (40.02%) had Aspergillus infection, 72 (15.58%) had Fusarium infection, 110 (23.08%) had Candida infection, 66 (14.28%) had Curvularia infection [Table 5].

Table 5 Details of clinical features and outcomes among four major groups of patients with fungal keratitis

Risk Factors	Aspergillus (n = 371)	Fusarium (n = 171)	Candida (n = 127)	Curvularia (n = 80)
Central opacification	279	117	78	65
Hypopyon	183	88	64	45
Duration of illness (days)	28.39 ± 11.24	32.56 ± 14.86	31.77 ± 15.66	40.83 ± 19.59 ^*
Therapeutic keratoplasty (Total – 462)	186	72	110	66
^* Statistically significant on comparing Curvularia with others (P = 0.046)

Mycotic keratitis frequently mimics as bacterial or parasitic keratitis, and it is very difficult to differentiate clinically. For delayed presentation and difficulty in diagnosis large number of patients do not get proper medication in time and present with delayed presentation. To estimate the load of fungal keratitis and evaluate the spectrum of causative organisms, their presentations, course and treatment and other epidemiological factors affecting the disease progression, we have conducted this study in our institute over 10 years.

Our study revealed that 38.92% of microbial keratitis is due to fungal by origin. This high incidence of fungal keratitis can also be seen in neighbouring area of same climatic region, like Bangladesh (39.9%) and Orissa (32%) [8,9]. Whereas incidence of mycotic keratitis in South India is higher, which is 44%, because of hotter and more humid climate than Eastern India, which favours the growth of fungus

Till date, globally, 56 genera and in India 27 genera had been reported for causing mycotic keratitis. But in our study, we had identified 32 different genera of fungus which had caused keratitis within last 10 years in Eastern India.

West Bengal has diverse climatic regions, and for this geographical variation reflects prominently in their incidence. Filamentous fungi were predominantly isolated in our study as was noted the same from the studies from tropical and subtropical countries, such as NepalBangladesh, Ghana [8,10,11]. Aspergillus (45.75%) and Fusarium (21.09%) were predominant causative organisms for mycotic keratitis in our study. Whereas Fusarium is predominant in South India and Aspergillus is predominant in rest of the India. Candida and other yeasts had been isolated mainly from the North Bengal region and Sub Himalayan part of West Bengal, Sikkim. The climate of this region matches with European countries where yeasts isolated predominantly [12]. Curvularia is found mostly in hot and humid area. It is reported that it is predominant along the US gulf coast of Mexico and in India it is predominant in North India [13]. In West Bengal it is predominant in Purulia, Bankura, Coast of Bay of Bengal in Midnapore and in South 24 Parganas for similar climate.

Seasonal variation on fungal keratitis had been analysed previously in other studies, where it had been observed fungal keratitis increases during the harvesting season and peaked in the post monsoon time. High humidity favours the growth of fungus. In our study similar seasonal variation is reflected.

A male predominance (62.15%) was seen in this study, which also corroborates other studies in India mostly due to the occupational causes. A wide range of age groups were presented to us, from 4 to 74 years of age. It had been noted that dependent age groups have presented mostly with accidental trauma by Finger nail injury, bird beak injury, cow horn/ tail injury etc. whereas the independent age groups patients were presented mostly with corneal injuries due to vegetative matter during agricultural season.

In this study trauma with vegetative matter is the most common risk factor for mycotic keratitis. History of trauma due to vegetative matter is seen in 37.46% in Aspergillus keratitis, 32.16% in Fusarium keratitis. Whereas Candida is mostly seen in immunocompromised patient (27.55%). This similar conclusion has been drawn by H Mohammad J Hassan et al [14].

The patients with dematiaceous fungal ulcer took higher time to resolve than the others, as it is chronic slow progressing non healing ulcer.

In our study, every patient received topical natamycin with or without other antifungal agents. Topical Natamycin suspension is alone effective for Aspergillus, but in resistant cases Oral Fluconazole have been used. In case of Fungal ball in Anterior chamber, Amphotericin B was given in Anterior chamber and for deep stromal infiltration stromal injection of voriconazole has been given.

Surgical intervention as therapeutic keratoplasty is an important mode of management. In our study, 43.03% patients showed response to medical therapy, while 56.96% of mycotic keratitis patients required therapeutic keratoplasty. Within this 40.02% of Aspergillus and 110 out of 127 Candida Keratitis required Keratoplasty. Regina L et al reported from Texas that out of 29 Candida keratitis patients, 15 patient (51.72%) required surgical intervention of which 13 patients had therapeutic penetrating keratoplasty and 2 eyes needed enucleation [15].

In conclusion, Aspergillus remains highest in number in incidence followed by Fusarium. It follows seasonal variation and post monsoon period is the peak season for fungal keratitis. Trauma with vegetative matter is the most common risk factor for the mycotic keratitis. Natamycin suspension can be the drug of choice but therapeutic keratoplasty can be the definitive treatment option in Mycotic Keratitis.

FUNDING: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

COMPETING INTERESTS: The authors have no relevant financial or non-financial interests to disclose. The authors have no competing interests to declare that are relevant to the content of this article.

AUTHOR CONTRIBUTIONS: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by [Ashis Majumder], [Jayanta Dutta] and [Sneha Jain]. The first draft of the manuscript was written by [Jayanta Dutta] and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

ETHICS APPROVAL: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Institutional Ethics Committee (West Bengal university of Health Sciences) on date:11^th Feb 2020

CONSENT TO PARTICIPATE: Informed consent was obtained from all individual participants included in the study.

CONSENT TO PUBLISH: The authors affirm that human research participants provided informed consent for publication of their personal information and data for this study

ACKNOWLEDGEMENT: We acknowledge the help of all clinical and non- clinical staffs of Regional Institute of Ophthalmology, Kolkata, West Bengal, India who helped us in successful conduct of this study

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Mycotic Keratitis in Eastern India: Report of a Decade

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