Download rajive gandhi university of health sciences, karnataka

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE – 41
REGISTRATION OF SUBJECT FOR DISSERTATION
1. NAME OF THE CANDIDATE AND SHIVARAJ,
ADDRESS
POST GRADUATE (M. Sc. M.L.T.),
DEPARTMENT OF MICROBIOLOGY,
ST. JOHN’S MEDICAL COLLEGE AND
HOSPITAL,
SARJAPUR ROAD,
BANGALORE-560034.
2. NAME OF THE INSTITUTION
ST. JOHNS MEDICAL COLLEGE
3. COURSE OF STUDY & SUBJECT
M. Sc. M.L.T. (MICROBIOLOGY)
4. DATE OF ADMISSION TO THE SEPT 1ST 2009
COURSE
5. TITLE
DETECTION,
ISOLATION
AND
SPECIATION OF MALASSEZIA FROM
PATIENTS
WITH
SUPERFICIAL
CUTANEOUS MYCOSES.
1
6.
Brief Resume of the study
6.1 Need for study:
Superficial cutaneous mycosis is a common clinical condition seen in our country.
It may be caused by Dermatophytes, Malassezia, Candida or Trichosporon species. The
genus Malassezia most commonly cause superficial cutaneous infection, but rarely in
people with certain predisposing factors become invasive and cause opportunistic
systemic infection1,2. Among the superficial cutaneous infections, Pityriasis versicolor is
the most common and others include seborrheic dermatitis, atopic dermatitis and
folliculitis2. The taxonomic status of this genus has been recently expanded to include
eleven species3,4.
Although diagnosis based on typical yellow fluorescence of the involved skin on
Wood’s Lamp examination and “spaghetti and meat ball” or “banana and grape”
appearance on direct microscopy with 10% Potassium hydroxide (KOH) examination of
skin scrapings are technically simple and offer quick diagnosis, culture based diagnosis is
of utmost importance to establish the clinico-mycological relationship2,5.
All species of Malassezia except Malassezia pachydermatis are lipophilic and
therefore their isolation and speciation, unlike other fungi, is technically demanding.
Although, there is evidence to suggest variation in geographical distribution of the
different species, not much data is available from our country4,5,6. This study, therefore,
will help us to establish and standardize techniques of isolation and speciation of
Malassezia and also understand the species distribution in our centre.
6.2 Review of literature:
The genus Malassezia is commonly responsible for a variety of superficial
cutaneous infections and rarely opportunistic systemic fungal infection in immunocompromised patients. Pityriasis versicolor is the most common presentation and the
others include seborrheic dermatitis, atopic dermatitis and folliculitis2,5.
The fungus that causes Pityriasis versicolor was detected and described for the
first time by Eichstedt in 1846 and by Sluyter in 18477. In 1853, Robin named the fungus
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as Microsporum furfur, as he thought it was similar to the dermatophyte,
Microsporum audouinii. Hence, the disease was renamed as tinea versicolor1. In 1874,
Malassez described yeast like cells from a lesion of the scalp and called them “Spores”.
In 1884, Bizozero observed spherical as well as elliptical yeast like cells in epidermal
scales of the humans and noted that these yeast cells were similar to the “Spores of
Malessez” and named spherical cells Sacchromyces sphaericus and the oval cells S.
ovalis. In 1889, Baillon recognized the etiological agent to be different from
Microsporum species and included this group of yeasts under a new genus Malessezia7.
In 1904, Sabouraud considered this organism as a causative agent of dandruff and gave it
a new name, Pityrosporum malassez1. Acton and Panja considered Pityrosporum to be
synonym of Malassezia, but they failed to prove it7. But in 1969, Sternberg and Keddie
detected the same antigenic components in Pityrosporum orbiculare and Malassezia
furfur by the fluorescent antibody technique which proved that both are synonymous1.
Isolation of the fungus in culture media was not successful until the lipophilic
nature of Malassezia was described in 1939 by Barham7. In 1951 Gordon isolated yeast
like fungus and named it as Pityrosporum orbiculare. In 1961, Burke was able to produce
clinical Pityriasis versicolor by inoculating Pityrosporum orbiculare on the skin of
persons with high plasma cortisol levels. In 1981, Redline and Dahm reported for the first
time invasion of deep tissue by M. furfur and later Hassall and colleagues reported the
same in young children receiving intravenous lipid therapy7.
The distribution of Malassezia infection is world wide, but the disease is more
prevalent in tropical and subtropical climates5,7. Studies from India show infection to be
twice more common in males. It occurs mostly in the age group of 11-20 years. The
disease is rare above 50 years of age8. Species distribution varies in different parts of
India. M. sympodialis is the predominant species of human skin, in health and disease,
followed by M. globosa, which is commonly found in scales of Pityriasis versicolor.
M. restricta seems to be associated with Pityriasis capitis6,9. M. furfur is seldom found as
normal flora or in disease states9.
Recently the taxonomic status of the genus Malassezia has been expanded to
include eleven species by their physio-biochemical properties as well as molecular
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studies3,4. It includes M. globosa, M. restricta, M. sympodialis, M. furfur, M. obtusa,
M. slooffiae, M. pachydermatis, M. japonica, M. nana, M. dermatis and M. yamatoensis.
Important techniques used in the diagnosis and identification of Malassezia
include, Wood’s Lamp examination of the lesions which show golden yellow
fluorescence of the scaly lesions. Direct microscopic examination of 10% KOH
preparation of skin scraping shows characteristic “spaghetti and meat balls” or “banana
and grape” appearance2,5. Addition of an equal amount of Parker blue/black Quink
permanent fountain ink to KOH (Parkers stain) enhances the visibility, as the bright blue
stain is taken up by the organism. Calcofluor white fluorescent staining may also be used
to avoid confusion from artifacts1,2,5.
Isolation of Malassezia from clinical samples is done by inoculation on
Sabouraud Dextrose Agar (SDA) with 0.5% chloramphenicol, 0.5% cycloheximide and
source of lipid, in the form of olive oil2,5. Colonies are characteristically small 3-6mm,
cream to yellow in colour, with slightly raised irregular edges, developing within 5-6
days at 32-35oC2,10. Lactophenol cotton blue (LPCB) mounting can be done for
characteristic microscopic features. Apart from S.D.A., other media such as mDixon
(modified Dixon) agar and Leeming and Notman agar can be used5.
Speciation of Malassezia is done by routine biochemical and physiological tests
like lipid dependency for their growth, temperature tolerance, catalase reaction, esculin
splitting by β-glucosidase activity and tween assimilation2,3,5. Recently molecular typing
methods such as Polymerase Chain Reaction (PCR) and Restriction fragment length
polymorphism (RFLP) have also been used in speciation3,5.
Malassezia species are susceptible to a variety of topical antifungal agents like
2.5% selenium sulphide, 1% zinc pyrithione and azoles such as clotrimazole and 2%
ketoconozole5,11. The treatment duration is about 2-3 weeks11. In case of failure with
topical agents or in severe cases with extensive lesions use of systemic antifungals with
oral ketoconozole, fluconozole or itracozole is found to be effective5,12. Patients with
fungemia can be treated with intravenous administration of amphotericin B or
fluconozole4.
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6.3 Objectives of the study:
1. To study the occurrence of Malassezia in clinically suspected cases of superficial
cutaneous mycoses.
2. To isolate and speciate Malassezia from the above cases.
3. To study the clinical correlation in patients with underlying disease conditions
from whom Malassezia is isolated
7. Materials and Methods:
7.1 Source of Data:
Patients visiting the Dermatology OPD of St. John’s Medical College and
Hospital during the period 1st January 2010 to 31st December 2010 whose skin
scrapings on direct KOH preparation reveal the typical “spaghetti and meat balls”
or “banana and grape” appearance will be included in this study. A minimum of
100 such clinical samples will be studied.
7.2 Methods:

Sample Collection2:
Skin scrapings will be obtained from the multiple sites of lesion after cleaning
the site with 70% alcohol using sterile blunt scalpel.

Sample processing2,5:
1. Direct microscopic examination of skin scrapings: using 10% KOH.
2. Culture: S.D.A with 0.5% chloramphenicol, 0.5% cycloheximide and
olive oil as lipid source will be used.
 Speciation of isolates: by their physio-biochemical properties using the
following tests2,3,5.
1. Lipid dependency for their growth.
2. Temperature tolerance.
3. Catalase reaction.
4. Esculin splitting by β-glucosidase activity.
5. Tween assimilation.
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 Data to be collected from medical records will include:
I. Demographic details of patient
Name
Age
Sex
Address
Occupation
II. Distribution of lesions and type (as described by the treating
dermatologist).
III. Wood’s Lamp Examination findings (as described by the treating
dermatologist).
IV. Underlying medical conditions such as Diabetes Mellitus, Steroid
therapy, HIV status (if report already available or test done as part of
treatment) and any other illness.
7.3 Inclusion Criteria:
Only those patients whose skin scrapings on direct 10% KOH examination
reveal the typical “spaghetti and meat balls” or “banana and grape” appearance
will be included in this study.
7.4 Exclusion criteria:
Those patients whose skin scrapings on direct microscopic examination do
not show the typical appearance are excluded from the study.
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
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8. References:
1. Inamadar Arun C, Palit Aparna. The genus Malassezia and human disease. Ind J
Dermatol Venereol Leprol 2003;69(4):265-70
2. Chander Jagadish. Medical Mycology. 2nd ed. New Delhi (India): Mehtha; 2002.
p. 70-78
3. Shokohi T, Afshar P, Barzgar A. Distribution of Malassezia species in patients
with Pityriasis versicolor in Northern Iran. Ind J Med Microbio 2009;27(4):32124.
4. Anaissie Elias J, Ginnis Michael Mc. R, Haller Michell AP. Clinical Mycology.
2nd ed. China: Churchill Livingstone Elesvier; 2009. p. 251-54
5. Merz Willium G, Hay Roderick J, editors. Topley & Wilsons Microbiology and
Microbial Infections – Medical Mycology. 10th ed. London (Eng): Hodder
Arnold; 2005. p. 185,202-19
6. Kindo AJ, Sophia SKC, Kalyani J, Anandan S. Identification of Malassezia
species. Ind J Med Microbio 2004;22(3):179-81.
7. K.J. Kwon – Chung, Bennett John E. Medical Mycology. USA: Lea & Febiger;
1992. p. 170-82.
8. Ghosh Sudip Kumar, Dey Sunil Kumar, Saha Indranil, Barbhuiya Jayashree Nath,
Ghosh
Arghyaprasun,
Roy
Aloke
Kumar.
Pityriasis
versicolor:
A
clinicomycological and epidemiological study from a tertiary care hospital. Ind J
Dermato 2008;53(4):182-5.
9. Dutta Sidhartha, Bajaj AK, Basu S, Anupam Dikshit. Pityriasis versicolor:
Socioeconomic and clinic-mycologic study in India. Int J of Dermato
2002;41:823-4
10. Larone Davise H. Medically Important Fungi – A guide to identification. 4th ed.
Washington(USA): ASM; 2002. p. 136-7,309
11. Cook Gordon. Mansons Tropical diseases. 20th ed. London (Eng): Saunders;
1996. p. 1055-56
12. Fauci Anthony S. Braunwald Eugene, Kasper Dennis L, Hauser Stephen L, Longo
L. Dan, Jameson Larry J et. al. editiors. Harrisons principles of internal medicine:
Vol 1. 17th ed. USA: The Mc-Graw Hill Medical; 2008. p. 1263
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SIGNATURE OF THE CANDIDATE
NAME AND DESIGNATION OF THE DR. JAYANTHI SAVIO,
ASSISTANT PROFESSOR,
GUIDE
DEPARTMENT OF MICROBIOLOGY,
ST. JOHN’S MEDICAL COLLEGE AND
HOSPITAL,
BANGALORE - 560034
REMARKS OF THE GUIDE
SIGNATURE OF THE GUIDE
DR. S. MURALIDHARAN
HEAD OF THE DEPARTMENT
PROFESSOR,
DEPT. OF MICROBILOGY,
ST. JOHN’S MEDICAL COLLEGE AND
HOSPITAL,
BANGALORE - 560034
SIGNATURE OF THE HOD
REMARKS
OF
THE
DEAN
OF
DEAN
OF
INSTITUTION
SIGNATURE
OF
THE
INSTITUTION
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