Rajiv Gandhi Universiry of Health Scences, Karnataka

RAJIV GANDHI UNIVERSIRY OF HEALTH SCENCES, KARNATAKA,

BANGALORE-41

PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION

6. BREIF RESUME OF THE STUDY

6.1 NEED FOR THE STUDY:

Fungi are ubiquitous in nature and exist as free organisms that derive no obvious benefits from parasitizing humans or animals.(1)

There has been a steady increase in the number of patients suffering from life threatening fungalinfections particularly among the immunocompromised individual. Underlying predisposing factors such as immunocompromised situations due to use of corticosteroids, antimicrobials,immunosuppressive anti cancer drugs, transplantation, metabolic disorder (i.e. diabetes mellitus) increased invasive procedures have been associated with increased fungal infections. There is a need for isolation and identification of fungi for prompt changes in treatment.(2)

6.2 REVIEW OF LITERATURE

There are three main groups of pathogenic fungi which are quite different from one another in the disease process. 1) Dermatophytes are a group of obligate fungal infection which attack human skin, nails and hair. 2) Dimorphic fungi are a group of soil borne fungi which have developed a different morphology in order to adapt to the hostile environment of the human body. These organisms can cause diseases in healthy people also. 3) Thethird group which is the most numerous consists of opportunistic fungi which can attack human whose immune systems are deficient in some way or artificially suppressed. (3)

The Dermotophytes are a distinct group of filamentous fungi that infectthe skin, hair and nails of human and animals, producing a variety of cutaneousmanifestations (4,5).The disease caused by Dermatophytes is known as Dermatophytosis. Their prevalence is governed by environmental conditions, personal hygiene and individual susceptibility. These are divided into three genera depending on their morphological characteristics. (2)

Most of the fungal species grow either as yeast or molds but some species are capable of growing in more than one form under different environmental conditions. They are known as dimorphic fungi. Their change in the somatic structure is governed principally by the temperature of incubation, yeast forms at 370 C and filamentousforms at 250 C. Most of the true pathogenic fungi are dimorphic in nature. (2)

The term opportunistic is used for any nonpathogenic fungi that can cause subcutaneous and disseminated infection. These are fungi, usually of inherent low or limited virulence, that nevertheless can cause local or disseminated disease in individuals who are debilitated, who are immunosuppressed, are who carry intravascular or prosthetic devices. (7,8,9,10)

1. fumigatus

1. flavus

1. niger

6.3 OBJECTIVES.

1)Isolation and Identification of filamentous fungi from clinical samples.

2)Comparison of microscopy with culture for detection of these fungi from clinical samples.

7. MERERIALS AND METHODS.

7.1 Source of study.

The study is to be carried out at the Department of microbiology St. Johns medical college Bangalore (Duration 2010- 2011).

7.2 METHODS

Samples to be collected

Nail, sputum, BAL, skin, tracheal aspirate, tissue, nasal swab, corneal scraping etc. sent to the microbiology laboratory will be tested.(11)

Samples processing.

All samples will be processed in a biosafety cabinet using standard procedure.

Fluid.

Sterile fluids will be centrifuged for 20 min at 1500- 2000 rpm and the sediment will be inoculated directly onto the surface of SDA media.

Tissue – when processing tissues for the recovery of fungi, the use of mortar and pestle or a tissue grinder should be avoided as the hyphal forms can easily be destroyed by grinding, making it difficult to recover viable organisms on culture. Mince the tissue into 1mm cubes with sterile scissors or a sharp scalpel blade and place the times fragments directly on to the agar submerging them slightly beneath the sample with an inoculating needle, mark on the reverse side of the inoculation.

Sample of tissue homogenate, bone marrow should be placed on the surface of SDA culture media. Non selective culture media, example BA, CA, without antibiotics are also adequate, as these specimens are usually sterile.

Skin scales, nail scraping or hair should be placed directly on the surface of the culture media such as SDA with chloramphenicol and cycloheximide with a straight inoculating wire, submerge a few fragments beneath the agar surface. Examine in the areas of inoculation at frequent intervals for the appearance of surface colonies.(7)

Corneal Scraping:

Scrapings will be inoculated at bedsideon Sabouraud’s Dextrose Agar (SDA) with chloramphenicol and scrapping materials will be cultured on blood agar (BA) and chocolate agar (CA). Inoculation will be made in the form of ‘C’ shaped streak. (12)

Direct Microscopy:

Potassium hydroxide (KOH) solutions.

KOH is used in two different concentrations to clear specimens that are cloudy or opaque so that the mycotic elements are visible. The stronger 20% solution is recommended for use with scrapping from fingernails or toenails. 10% KOH is used for all other specimens. (13)

In this procedure KOH is mixed in equal proportion with the specimen on a slide with a coverslip over it and left for 5 -10 minute for digestion. Any fungi present will be visible as KOH dissolves keratin and other cellular material in the specimen and express the fungal filaments as refractile filaments (septate or a septate). (14)

Culture Media:

Selection of culture media for the isolation of organism depends on the type of fungi. Most commonly used culture media is SDA (Sabourads dextrose Agar) with or without antimicrobial agents. eg: for the isolation of dermatophytes SDA with chloramphenicol and cycloheximide is used whereas for the isolation of saprophytic opportunistic fungi SDA with chloramphenicol and without cycloheximide is used, as cycloheximide inhibits the growth of saprophytic fungi.

Two sets of inoculated tube will be incubated, one at room temperature (i.e. 250C) and the other at 370C

All the culture tubes should be examined for growth daily for a week and twice a week for further three week before being considered negative. Further any growth will be identified by microscopy (LPCB mount) and slide culture will be put to identify the characteristic features.

Attempt will be made to identify dermatophytes to species level. The additional tests required are.

1. Dermatophyte test medium(DTM) - at 250C is used to isolate and distinguish dermatophytes from the fungal or bacterial contaminant found in cutaneous lesions.
2. Hair perforation test – is done to differentiate betweenT.mentagrophytes, T.rubrum,M.canis and M.equinum respectively. (2)

Differentiation of similar conidia producing Trichophytm spp

Abbreviations + positive, O negative, w weaks + trace, 4 + maximum growth, V variable. (11)

7.3Inclusion criteria

All samples that are received for fungal cultures in the microbiology laboratory.

7.4 Exclusion criteria.

All samples in which yeast is grown or demonstrated by microcopy will be excluded (including dimorphic fungi).

7.5 Does the study require any investigation or interventions to be conducted on patients or other than human beings or animals? If so, describe briefly.

No

7.6 Has ethical clearance been obtained from your institution in case of 7.3

Not applicable

References

1. George.S.Kobayashi : Disease mechanism of Fungi, 4th Edition; Copyright © 1996, The University of Texas Medical Branch at Galveston
2. Chander J: A textbook of Medical Microbiology 1st edition 1996, p.3,6,7,18,67-79,149-154,155-161.
3. Development of fungal taxanomy clinical Microbiology review 1999:12: 454-500.
4. Singh S, Beena PM. Comparative study of different microscopic techniques and culture media for the isolation of Dermatophytes. Indian Journal of Medical Microbiology 2003;21(1):21-4
5. Kaur R, Kashyap B, Bhalla P. Onychomycosis - Epidemiology, diagnosis and management. Indian Journal of Medical Microbiology 2008;26(2):108-16.
6. Kwon Chung, John E Beneett, Medical Mycology copyright @ 1992 by Lea and Febiger, p107.
7. Koneman Diagnostic Microbiology, 5th edition Lippincott 1997 p.983-1057.
8. Anuradha K, Lakshmi V, Umabala P, Rao MN- Pulmonary Zygomycosis in diabetic patient.Indian Journal of Medical Microbiology 2006;24(3):222-24
9. Tuzcu A, Bahceci M, Celen MK, Kilinc N, Ozmen S. Necrotizing (malignant) otitis externa: An usual localization of mucormycosis. Indian Journal of Medical Microbiology 2006;24(4):289-91
10. Mohapathra S, Xess I, Swetha JV, Tanveer N, Asati D, Ramam M et. al. Primary cutaneous aspergillosis due to aspergillus niger in an immunocompetent patient. Indian Journal of Medical Microbiology 2009;27(4):367-70
11. Davise H. Larone, Medically important fungi, A guide to identification 4th edition, ASM 2002 p.45-51, 242-297.
12. Asha Pichare,Neeta Patwardhan,Damle AS,Deshmukh AB :Bacteriological and mycological study of corneal ulcers in and around Aurangabad. Indian J. Pathol Micro 2004:47 (2): 284-286.
13. Frank Fisher, Fundamentals of diagnostic mycology, 1st edition (1998) p.336.
14. Connie R Mahon, Donald. C.Lehman, George Menuselis.Text book of diagnostic microbiology. 2nd edition p.718.