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Isolation of fungi from corneal ulcers in patients attending tertiary care hospital.

INTRODUCTION: In the tropical, agricultural, and developing countries like India, fungal infection of eye is one of the major problems. It is not uncommon to find infection of cornea with varied fungi of soil in South India. It has been observed that conjunctival flora vary according to the environment and population. Fungi, with few exceptions, are essentially free living saprophytes in nature. Fungal diseases are usually not contagious. Mycotic infections in man and animals follow traumatic implantation of the fungi as commensals and are capable of causing endogenous disease under special circumstances like lowered immunity, metabolic disorders and malignancy etc. (1)

The cornea, outer most and transparent coat of the eye, plays a vital role for vision, the most precious gift of the nature. Cornea, being always exposed to the environment during the most of the working hours. Many a time, the injurious material carries diverse infective agents like bacteria, viruses and fungi along with it. In most occasions; the minor trauma occurring in day to day life heals by itself due to normal repairing mechanisms of the body. But it some cases, when the body's resistance is lowered or the dose and virulence of the microbial agents are more, even a minor injury can result in the formation of corneal ulcer.

In our country, such infections have been one of the main problems. In the last 4 decades, however, the incidence of fungal infection of the eye mostly corneal, has increased considerably, a phenomenon which seems to be due to two factors:

1. An upset of normal symbiosis between bacteria and fungi since the introduction of antibiotics into eye and.

2. An alteration of the resistance of the tissue from the use of corticosteroids.

These two factors allow saprophytic fungi to become facultative pathogens.

The importance of early diagnosis of Keratomycosis is stressed, for the eyes not treated in time, may lead to complications and blindness. The microbiologist should be aware of fungi as possible pathogenic agents whenever the material is processed from eye infection. Increase in the incidence of Keratomycosis during the past few years has been due to better diagnostic methods and the recognition that fungi which have been considered as non-pathogenic do have the ability to infect eye. Furthermore, absolute increase in the number of fungal infections has probably resulted from the widespread and indiscriminate use of antibiotics and corticosteroids in ocular therapy also. As the line of treatment in fungal ocular lesions is different from that of bacterial infections, it is felt that detection of diseases like mycotic keratitis is important. It will go a long way in the formulation of therapy. Hence, the present study is made to assess the incidence of fungi that causes keratitis in Rayalaseema area.

MATERIAL AND METHODS: 102 patients with corneal ulcer were analyzed for fungi, as aetiological agents. A control group was included, comprising of 100 healthy human beings from whom, conjunctival swabs were collected and analyzed.

Cornea was anaesthetized with topical 4% lignocaine and exposed by universal speculum. Corneal scrapings were taken from margin and base of the ulcer and processed as follows:

A. DIRECT WET MOUNT PREPARATION, WHICH INCLUDED:

I. KOH wet mount preparation.

II. Lacto Phenol Cotton Blue, wet mount preparation.

III. GRAM's staining.

KOH WET MOUNT PREPARATION:

i. A portion of scraped material was transferred on a sterile glass slide and a drop of 10% Potassium Hydroxide was added to it. A cover slip was placed on it and left undisturbed for 10 to 15 min. then it was examined under low power and high power objective. (2) Lacto Phenol Cotton Blue, wet mount preparation.

LACTO PHENOL COTTON BLUE WET MOUNT PREPARATION: A drop of stain was placed on a clean glass slide. The scrapings from the ulcer were placed in the stain and mixed. A cover slip was placed over the mixture with little pressure avoiding air bubbling formation. Then it was observed under low power and high power objective. (2, 3)

GRAM STAINING (HUCKER'S MODIFICATION): Hucker's Modification of Gram staining was followed where crystal violet was used as primary staining.

B. CULTURE FOR FUNGI IN SABORAUD'S DEXTROSE AGAR MEDIUM: Sabouraud's dextrose agar medium was used for isolation of fungi. The material was inoculated into four slants of Sabouraud's dextrose agar medium, two with chloramphenicol and two without chloramphenicol. Two slants were incubated at room temperature and other two slants incubated at 37[degrees]c.

An uninoculated medium was exposed by the side of the eye for few seconds and incubated along with the inoculated media as control. The inoculated slants were observed daily for evidence of growth. Earliest growth of fungus appeared within one week but slants were kept for four weeks before being discarded as negative. The growths from slants were identified by their cultural characters and microscopic examination.

In case of Candida, the following procedures were carried out to identify the species:

1. Grams Staining.

2. Germ Tube Formation.

3. Chlamydospore Formation.

4. Sugar Fermentation Tests.

SLIDE CULTURE TECHNIQUE: Once growth was observed on Saboaraud's dextrose agar slide culture technique was followed for more detailed morphological identification of fungi.

A slide was placed on a bent (U shaped) glass rod in the bottom of a Petridish, covered and sterilized. Saboauraud dextrose agar prepared in plates of about 15 ml of agar per plate was allowed to solidify. Blocks of about 1 cm agar per plate was allowed to solidify. Blocks of about 1 cm square and 2-3 mm depth were taken out from the agar plate using all sterile techniques and placed on the sterile slide in Petridish. Centers of four sides of agar block are inoculated with the culture. The inoculated block was covered with a sterile cover slip. 8 cc of sterile water is added to the bottom of sterile Petridish, covered and incubated at room temperature. This was examined periodically for growth. If there was evaporation of water from Petridish, water was added again and further incubated until growth was observed. (2)

When sporulation was well developed, the cover slip was lifted completely and placed on a drop of lacto phenol cotton blue and observed under microscope.

RESULTS: Corneal scrapings, from 102 cases suffering from corneal ulcer and conjunctival swabs from 100 healthy persons were processed for fungi. Former group constituted the study group, and the latter control group.

In the study group, direct microscopy revealed fungi in 18 cases, and culture yielded fungi in 28 cases. In 74 cases culture yielded no growth.

In this study group, fungal elements were seen in 18 cases. All the three i.e. KOH, LPCB, GRAMS showed positive for fungi in 16 cases, in 2 cases KOH only showed fungal elements.

In this study group, Asperigillus was the predominant causative agent of corneal ulcer (39.28%) followed by Fusarium (17.85%), Curvularia (10.71%), Candida (7.14%), Cephalosproium (7.14%), Rhizopus (3.57%), Pencillium (3.57%), Mucor (3.57%) and Unidentified (7.14%), as shown in the above table.

In the study group, Aspergillus flavus was the predominant causative agent (63.63%) followed by Aspergillus fumigates (27.27%), and Aspergillus Niger (9.09%).

A high incidence of 36.84% was noted in the age of group of 41-50 yrs in males and in the females 28.57% was noted in the age group of 51-60 yrs.

History suggestive of trauma was the leading predisposing factor in the present study (53.57%) followed by local and systemic use of antibiotics and corticosteroids (21.42%).

The incidence of Kerato mycosis was highest in the agricultural workers in the present study with an incidence of 57%.

A high incidence of 38.09%was noted in the month of October, followed by September with an incidence of 35.71%.

100 conjunctival swabs were collected from control group, processed for fungi.

In the control group, culture yielded growth in 8 cases (8%). 5 of males and 3 of females.

DISCUSSION: In the present study, among 28 mycotic corneal ulcers, Aspergillus species were isolated in 11 cases with an incidence of 39.28%, in 7 cases Aspergillus flavus was isolated, followed by fumigates in 3 cases and in one case Aspergillus niger was isolated.

Many workers in their studies stated that Aspergillus species were the predominant causative agents. Subbannayya from Manipal (4) 34%, Porias Dogre (5) 27%, Anitha Panda (6) New Delhi 39.5% and savitri sharma (7)

In the present study, Fusarium species were isolated in 5 cases (17%), Candida in 2 cases, Rhizopus, Penicillium and Mucor in each one case and in 3 cases fungi were not identified.

In the present study, Fusarium species were isolated in 5 cases with an incidence of 17%. Similar studies by several workers revealed an overall incidence of ranging from 8.5% to 32.3%.

In the present study, curvularia species were isolated in 3 cases from 28 with an incidence of 10.7%, this coincide with Anitha Panda 6 from Delhi (7.9%).

An interesting feature in the present study was Candida, which were isolated in two cases with an incidence of 7.14%. This also coincides with Siva Reddy, (8) PoriaDogre, (5) Anitha Panda (6) and other earlier workers. Candida had not been reported as a major corneal pathogen in most studies in tropical countries (1).

In the present study, Cephalosporium species are isolated in two cases with an incidence of 7.14%. Sunitha and Deshpande (9) with 2.33% and Anitha Panda with 3.7%. (6)

The incidence of keratomycosis in the present study was more in males than in females. Males are affected in 19 cases with an incidence of 67% and females were affected in 9 cases with an incidence of 33%.

Trauma plays an important role in keratomycosis. In the present study, 15 cases were due to trauma of eye. In the present study 53.57% cases were associated with trauma. Dasgupta et al (10) with 54%, Siva Reddy (8) et al. 52.8%, Anitha Panda (6) with 55.3%, Taiwar and sahgal, (11) Zimmermann (12)

In the present study trauma of the eye was usually due to vegetable matter like paddy seeds, wooden stick of plants, thorn, lash of cow tail, falling sand. Among 28 patients, 8 cases (25.57%) were due to drugs used locally (antibiotics and corticosteroids) (13) for more than a month.

Out of 28 patients of keratomycosis, the incidence was more common in the rural area when compared to the urban area. 19 cases from rural areas and 9 cases from Urban were reported. The people in the rural area were mainly agricultural workers and these were the people who used drugs indiscriminately.

In the present study, large number of patients was agricultural workers with an incidence of 57.14%.

There was a seasonal variation of mycotic ulcers according to the climatic conditions. (14) In the present study the maximum incidence was mainly in cool and humid months. A high incidence of 38.09% was noted in the month of October followed by September with an incidence of 35.7%. Lower incidence was seen in November, December, January, February, March and April. Least incidence was noticed in May and June. From July to October 20 cases were reported out of 28 cases. This was due to the harvesting season. Various factors like humidity, temperature and rush of field work may account this seasonal variation.

CONTROL GROUP: In the control group fungi were isolated in 8 cases from a total of 100 cases with an incidence of 8%.

Similar studies by several workers revealed an overall incidence ranging from 6.7% to 18.4% in India, Sinha and Das (=15) 13.4%.

CONCLUSION: Mycotic keratitis can easily be confused with other forms of microbial keratitis. In order to save the vision, mycotic keratitis should be suspected in every patient with corneal ulcers and should be ruled out by proper laboratory methods before commencing steroids and antibiotic treatment.

Aspergillus species were observed to be one of the predominant organisms in air and hence, it can easily infect injured cornea.

Lack of a broad spectrum antifungal medicine for ophthalmic use was a depressing observation. It is our sincere desire that more research be conducted to discover effective antifungal medicine for ophthalmic use. Steps to improve ophthalmic care and treatment in the periphery and rural areas should also be taken to lower the incidence of keratomycosis along with other ophthalmic diseases.

DOI: 10.14260/jemds/2015/91

REFERENCES:

(1.) Thomas P.A. et al--Mycotic keratitis--an under estimated mycosis. Journal of Medical and veterinary mycology (1994) 32, 235-256.

(2.) Cruickshank, Diguid, MarmionSwaion, Medical Microbiology Text Book, 1975, No.5.

(3.) Baily Sell and Scott's 7th ed. Diagnostic Microbiology.

(4.) Subbannayya Kotigadde- mycotic keratitis study in coastal Karnataka. Ind. J. of Opthal, jan-march 1992; 40: 31-33.

(5.) Poria and Dogre-Study of mycotic keratitis in Jamnagar, Indian Journal of Opthal, July, 1985, 33: 229-231.

(6.) Panda A, Mohan M and Mukherjee G.-A histo pathological study of corneal buttons. Indian Journal of Opthal., September, 1984, 32:311-315.

(7.) Savitri Sharma--Fungal keratitis--Chief Microbiologist, L. V. Prasad Eye Institute, Hyderabad.

(8.) P. Siva Reddy, Satyendran, Satapathy and Kumar and P.R.R. study of normal conjunctival flora (Bacterial and fungal) in relation to ocular infection. Indian Journal of Opthal.,20:101 (1972)

(9.) A Sunitha and Deshpande--A study of Mycotic keratitis in Mumbai, Indian Journal of Pathology and Microbiology, 42 (1): 81-87, 1999.

(10.) Das Gupta, Roy, Saunder raj, Ramamurthy and lamba. Mycological studies in keratitis. Indian journal of medical research 61:169, 1972.

(11.) Talwar, P., Sehgal, S. C. Mycotic keratitis. Indian Journal of Medical research, 1978.

(12.) Zimmerman, L. E. Achieves of pathology Cicago, 50:591, 1950(quoted)survey of keratomycosis.

(13.) Agarwal, L. P., and Khosla, Mycotic Keratitis. Journal of All India Opthalmological Society, 1966;15(10).

(14.) Srivastava, O.P. Koul, R.L. and Gupta, Ind. J, of Medical Research, 1961.

(15.) Sinha, B.N., and Das, M. S. Fungal flora of conjunctiva in healthy and diseased eyes. Ind. J. of Medical Association, 51, 217, 1968.

P. Venkateswarlu [1], B. Nagajyothi [2], K. Anjaneyulu [3], B. Shanti Reddy [4]

AUTHORS:

[1.] P. Venkateswarlu

[2.] B. Nagajyothi

[3.] K. Anjaneyulu

[4.] B. Shanti Reddy

PARTICULARS OF CONTRIBUTORS:

[1.] Associate Professor, Department of Microbiology, Government Medical College, Anantapuramu.

[2.] Associate Professor, Department of Microbiology, Government Medical College, Anantapuramu.

[3.] Assistant Professor, Department of Ophthalmology, Government Regional Eye Hospital, Kurnool.

[4.] Associate Professor, Department of Microbiology, Government Medical College, Anantapuramu.

NAME ADDRESS EMAIL ID OF THE CORRESPONDING AUTHOR:

Dr. P. Venkateswarlu, Associate Professor, Department of Microbiology, Government Medical College, Anantapuramu.

E-mail: oshomicro@gmail.com

Date of Submission: 05/01/2015.

Date of Peer Review: 06/01/2015.

Date of Acceptance: 07/01/2015.

Date of Publishing: 09/01/2015.

TABLE 1: STUDY GROUP

Group     Males   Females    Direct      Culture positive    Culture
                            Microscopy                      Negative
                                          Males   Females

Study      63        39         18          19        9         74
(102)

Control    50        50         --           5        3         92
(100)

TABLE 2: DIRECT MICROSCOPY FINDINGS

 KOH    Positive    KOH    Positive    KOH    Negative
LPCB    Positive   LPCB    Negative   LPCB    Negative
GRAM    Positive   GRAM    Negative   GRAM    Negative
           16                 2                  10

TABLE 3: FUNGI ISOLATED

SL.NO.   FUNGI              NO. OF    PERCENTAGE
                             CASES        (%)

1.       Aspergillussp        11         39.28%
2.       Fusariumsp            5         17.85
3.       Curvulariasp          3         10.71%
4.       Candida sp            2         7.14%
5.       Cephalosporiumsp      2         7.14%
6.       Rhizopus              1         3.57%
7.       Penicilliumsp         1         3.57%
8.       Mucorsp               1         3.57%
9.       Unidentified          2         7.14%
         Total:               28        100.00%

TABLE 4: CONTROL GROUP

Total cases   Male   Female   Culture positive

                               Male     Female

100           50     50       5 (10%)   3 (6%)

TABLE 5: FUNGI ISOLATED FROM CONTROL GROUP

Fungi           Males   Females

Aspergillussp   3       1
Penicilliumsp   1       1
Rhizopussp      1       0
Candida sp      0       1
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Title Annotation:ORIGINAL ARTICLE
Author:Venkateswarlu, P.; Nagajyothi, B.; Anjaneyulu, K.; Reddy, B. Shanti
Publication:Journal of Evolution of Medical and Dental Sciences
Article Type:Report
Date:Jan 12, 2015
Words:2613
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