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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 56  |  Issue : 4  |  Page : 219-221

Clinico-microbial profile of fungal keratitis and detection of Pythium insidiosum: A pilot study


1 JIPMER, Puducherry, India
2 Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India
3 Department of Ophthalmology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Web Publication19-Feb-2019

Correspondence Address:
Prasoon Garg
Room Number 209, Osler House, New Hostel Complex, JIPMER, Puducherry
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/tjosr.tjosr_84_18

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  Abstract 


Background: Infectious keratitis is a leading cause of blindness, more so in developing countries where rates of corneal ulceration are much more than those of industrialized countries. The organisms responsible for infectious keratitis differ regionally, with bacterial organisms more common in temperate climates and fungal organisms more common in tropical climates. A large fraction of infectious corneal ulcers that are referred to tertiary care centers in South India is fungal in etiology. Methods: Parameters studied were age, sex, residential district, occupation, risk factor, clinical presentation, microscopic examination of the sample, fungal culture, and Pythium insidiosum. Results: History of trauma was observed in half the cases. History of contact lens usage or diabetes mellitus was not seen in any case. Hypopyon was seen in 50% of the cases of which 33.33% were immobile. KOH preparation for all the samples was negative. Of the 15 samples sent for culture, 12 grew no fungus and 2 were contaminated. One fungus grew and was identified as Aspergillus terreus. P. insidiosum could not be isolated.

Keywords: Aspergillus terreus, keratitis, Pythium insidiosum


How to cite this article:
Garg P, Singh R, Babu R. Clinico-microbial profile of fungal keratitis and detection of Pythium insidiosum: A pilot study. TNOA J Ophthalmic Sci Res 2018;56:219-21

How to cite this URL:
Garg P, Singh R, Babu R. Clinico-microbial profile of fungal keratitis and detection of Pythium insidiosum: A pilot study. TNOA J Ophthalmic Sci Res [serial online] 2018 [cited 2019 Mar 24];56:219-21. Available from: http://www.tnoajosr.com/text.asp?2018/56/4/219/252496




  Introduction Top


Infectious keratitis is a leading cause of blindness, more so in developing countries where rates of corneal ulceration are much more than those of industrialized countries.[1] The organisms responsible for infectious keratitis differ regionally, with bacterial organisms more common in temperate climates and fungal organisms more common in tropical climates. A large fraction of infectious corneal ulcers that are referred to tertiary care centers in South India is fungal in etiology. The patient population, geographical region, and prevailing socioeconomic conditions cause significant variations in the etiological and epidemiological pattern of corneal ulceration. Srinivasan et al. from South India reported that 44% of all central corneal ulcers were caused by fungi.[2] More than 70 species of filamentous fungi have been identified as the etiological agents of fungal keratitis. The most common fungal causes of infectious keratitis were Fusarium spp. (14.5% of cultures) and Aspergillus spp. (8.8%).[3] Early diagnosis and treatment of fungal keratitis are important in preventing complications and loss of vision. Pythium insidiosum is recently recognized agent of keratitis. It is a cosmopolitan, aquatic, fungus-likeorganism.[4] It is classified as an oomycete and placed in the kingdom Stramenopila. It lacks ergosterol and chitin.[4],[5],[6],[7] Many studies have reported 10%–23% of fungal isolates from patients with fungal keratitis as unidentified because of lack of sporulation in culture.[8] Although rarely reported, P. insidiosum may not be a rare cause of fungal keratitis but an under-reported one.[8],[9] The majorchallenge in management lies in the fact that it is misdiagnosed and treated as a fungal infection, because of its morphological resemblance to fungi and also because of the lack of specific diagnostic methods.[6],[10]

Objectives

  1. To determine the clinical profile of fungal keratitis
  2. To determine the microbiological profile of fungal keratitis
  3. To determine the proportion of P. insidiosum as a causative agent of keratitis.



  Methodology Top


Study design

The study design was a descriptive study.

Data collection period

After Institute ethical clearance till July 15, 2018.

Study participants

  1. Inclusion criteria: Suspected cases of fungal keratitis presented at the Ophthalmology Department of Jawaharlal Institute of Postgraduate Institute of Medical Education and Research during the study period.
  2. Exclusion criteria: Nil
  3. Number of groups to be studied: One.


Sampling

  1. Sampling size calculation: All patients presenting to the ophthalmology department during the study period were included in the study as per the inclusion criteria
  2. Sampling technique: Convenience sampling.


Study procedure

Institute ethical clearance was taken. Patients presenting in the ophthalmology department as per the inclusion criteria were prospectively enrolled in the study. The clinical presentations were recorded in a prestructured proforma.

Corneal scrapings were obtained with the help of the ophthalmology department. A part of the specimen was placed on a sterile, clean glass slide and a drop of 10% KOH was added. The slide was examined microscopically. Another part of the sample was plated on Sabouraud's Dextrose Agar (SDA) and blood agar plates. Site of inoculation was marked. SDA plates were incubated at 25°C and blood agar plates at 37°C for 1 week. Plates were examined daily for any growth. Molds were identified morphologically by lactophenol cotton blue mount. Yeasts were identified by morphology on corn meal agar and color on chromogenic media. Suspected Pythium growth on blood agar plate would be attempted for zoospores production using carnation leaf method if isolated, after incubating it in an induction medium for 24 h (Modified method as described by Sharma et al.).[5]

If clinical trial, whether registration with clinical trials registry-India will be done

Not applicable.

Parameters to be studied

Age, sex, residential district, occupation, risk factor, clinical presentation, microscopic examination of the sample, fungal culture and P. insidiosum.

Statistical tests to be used for data analysis

The distribution of data on categorical variables such as sex, residential district, occupation, risk factor, clinical presentation, microscopic examination of the sample, fungal culture, and P. insidiosum was expressed as frequency and percentages. The continuous variables like age were expressed in terms of mean with standard deviation or median with range. All statistical analysis was carried out at 5% level of significance and P < 0.05 was considered as statistically significant.

Ethical considerations

The study was approved by the JIPMER ethical committee with waiver of consent as the samples collected were any way indicated in routine and routine procedure was followed. No additional sample was collected for the study.


  Results Top


The study was conducted for 7 weeks from May 28, 2018 to July 15, 2018, during which only 15 cases of suspected fungal keratitis were enrolled. The mean age of the patients was 45 years with a standard deviation of 18.09 and median age was 44 years with a range between 17 and 77 years. Nine cases (60%) were male while 6 (40%) were female. There were no cases from Puducherry district and all cases were resident of different district of Tamil Nadu. About 66.66% of cases were employed as laborers, while the remaining were in agriculture. History of trauma was observed in half the cases. History of contact lens usage or diabetes mellitus was not seen in any case. Hypopyon was seen in 50% of the cases of which 33.33% were immobile. KOH preparation for all the samples was negative. Of the 15 samples sent for culture, 12 grew no fungus and 2 were contaminated. One fungus grew as cinnamon brown, velvety, and powdery growth with yellow reverse [Figure 1] and [Figure 2]. Lactophenol cotton blue mount preparation [Figure 3] of the fungal growth showed hyaline septate hyphae, smooth conidiophore, dome-shaped vesicle, metulae and phialides arranged in upper two-third of the vesical and round, smooth short chain of conidia. It was morphologically identified as Aspergillus terreus. Thus, the percentage of positive growth was 6.66%. P. insidiosum could not be isolated. The association of the demographic factors or risk factors with fungal culture was not carried out due to single culture isolate.
Figure 1: Culture of Aspergillus terreus (front view)

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Figure 2: Culture of Aspergillus terreus (back view)

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Figure 3: Lactophenol cotton blue mount preparation of Aspergillus terreus

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  Discussion Top


It has been observed that 15 cases of fungal keratitis presented in 7 weeks duration, which accounts for approximately 111 cases in a year. The annual incidence of infectious keratitis in a study by Lin et al. in a tertiary eye care center in South India was found to be 2322 of which about 636 were of confirmed fungal etiology.[11] A study by Punia et al. showed that females were less commonly affected than males, which is usually the case, and that 41–60 years was the most common age group of the disease.[12] Males are more involved in outdoor activities as compared to females. This data corresponds to the results of this study. The same study found that the most common predisposing factor was trauma to the cornea seen in 59.09% cases and that use of contact lens was not seen in any case, which is in accordance with our results. Hypopyon was seen in 40% of the cases. Nearly 39.8% smears were positive for fungus and 60.2% were negative in cases of microbial keratitis in the study by Rathi et al. and 67.27% of samples were culture positive fungal keratitis in a separate study.[13],[14] These results are in a stark contrast to those of this study. It is probably due to low sample size. The most common fungal causes of infectious keratitis were Fusarium spp. (14.5% of cultures) and Aspergillus spp. (8.8%).[3] Here only single isolate of A. terreus was detected. Very limited data are available on fungal keratitis caused by this organism. The prevalence of P. insidiosum as an etiological agent of fungal keratitis varied between 5.5% and 3.9% according to the study by Sharma et al. while we were unable to isolate the organism.[5] Long duration of the study and multicentric study may be planned to get the better microbiological profile of fungal keratitis.


  Conclusion Top


This study determined the clinical and microbiological profile of patients of infectious keratitis suspected of fungal etiology who presented at the outpatient department of the Jawaharlal Institute of Postgraduate Institute of Medical Education and Research. While most clinical findings were in accordance to the results obtained by other similar studies, microbiological findings were considerably low. A. terreus was only isolated as causative agent of fungal keratitis. There was no case of P. insidiosum detected during the study period.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Whitcher JP, Srinivasan M. Corneal ulceration in the developing world – A silent epidemic. Br J Ophthalmol 1997;81:622-3.  Back to cited text no. 1
    
2.
Srinivasan M, Gonzales CA, George C, Cevallos V, Mascarenhas JM, Asokan B, et al. Epidemiology and aetiological diagnosis of corneal ulceration in Madurai, South India. Br J Ophthalmol 1997;81:965-71.  Back to cited text no. 2
    
3.
Lalitha P, Prajna NV, Manoharan G, Srinivasan M, Mascarenhas J, Das M, et al. Trends in bacterial and fungal keratitis in South India, 2002-2012. Br J Ophthalmol 2015;99:192-4.  Back to cited text no. 3
    
4.
Barequet IS, Lavinsky F, Rosner M. Long-term follow-up after successful treatment of Pythium insidiosum keratitis in Israel. Semin Ophthalmol 2013;28:247-50.  Back to cited text no. 4
    
5.
Sharma S, Balne PK, Motukupally SR, Das S, Garg P, Sahu SK, et al. Pythium Insidiosum keratitis: Clinical profile and role of DNA sequencing and zoospore formation in diagnosis. Cornea 2015;34:438-42.  Back to cited text no. 5
    
6.
Mittal R, Jena SK, Desai A, Agarwal S. Pythium insidiosum keratitis: Histopathology and rapid novel diagnostic staining technique. Cornea 2017;36:1124-32.  Back to cited text no. 6
    
7.
Mendoza L, Vilela R. The mammalian pathogenic oomycetes. Curr Fungal Infect Rep 2013;7:198-208.  Back to cited text no. 7
    
8.
Thomas PA. Current perspectives on ophthalmic mycoses. Clin Microbiol Rev 2003;16:730-97.  Back to cited text no. 8
    
9.
Krajaejun T, Sathapatayavongs B, Pracharktam R, Nitiyanant P, Leelachaikul P, Wanachiwanawin W, et al. Clinical and epidemiological analyses of human pythiosis in Thailand. Clin Infect Dis 2006;43:569-76.  Back to cited text no. 9
    
10.
Ramappa M, Nagpal R, Sharma S, Chaurasia S. Successful medical management of presumptive Pythium insidiosum keratitis. Cornea 2017;36:511-4.  Back to cited text no. 10
    
11.
Lin CC, Lalitha P, Srinivasan M, Prajna NV, McLeod SD, Acharya NR, et al. Seasonal trends of microbial keratitis in South India. Cornea 2012;31:1123-7.  Back to cited text no. 11
    
12.
Punia RS, Kundu R, Chander J, Arya SK, Handa U, Mohan H, et al. Spectrum of fungal keratitis: Clinicopathologic study of 44 cases. Int J Ophthalmol 2014;7:114-7.  Back to cited text no. 12
    
13.
Rathi VM, Thakur M, Sharma S, Khanna R, Garg P. KOH mount as an aid in the management of infectious keratitis at secondary eye care centre. Br J Ophthalmol 2017;101:1447-50.  Back to cited text no. 13
    
14.
Saha S, Banerjee D, Khetan A, Sengupta J. Epidemiological profile of fungal keratitis in urban population of West Bengal, India. Oman J Ophthalmol 2009;2:114-8.  Back to cited text no. 14
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