INTRODUCTION

     Malassezia, a lipophilic yeast like fungus is a natural habitat of stratum corneum and has been implicated in the pathogenesis of a variety of opportunistic dermatological and systemic infections.  But studies show that most patients did not respond to standard treatment and presented with recurrence of infection. 
          Considering the importance of Malassezia in medical mycology, this study was performed to identify various Malassezia species affecting the population under stud




OBJECTIVES

Epidemiology - To know the age, sex and site of prevalence of Malassezia
infection.

Anti-fungal susceptibility -To identify the most effective anti-fungal drugs
to treat infections caused by Malassezia and to obtain their MIC (minimum
inhibitory concentration) values.

Molecular study -To do DNA extraction, Polymerase chain reaction (PCR)
and sequence the extracted genome, thereby identifying and speciation of
Malassezia.

MATERIALS AND METHODS

Malassezia species were isolated from the skin scrapping’s of the patients
with hypo/hyper pigmented lesions in the Dermatology OPD of our tertiary
care hospital for a period of two years from July 2016 to Feb 2018. Microscopic
Examination with 10% KOH was done. KOH positive samples were
cultured on Modified Dixons agar (MDA) and Sabouraud’s Dextrose Agar
(SDA) with olive oil overlay and incubated at 32oC. Gram stain, Catalase
and Urease test was performed. Stock cultures were prepared and
refrigerated.

Anti-fungal susceptibility- was done by broth Micro- dilution method in
accordance with CLSI M27-A3 guidelines. Since Malassezia are lipid
dependant yeast,To RPMI 40 ADD :-

GLUCOSE-1.8% PEPTONE-1% OX BILE-0.5% GLYCEROL-1%
TWEEN 80-0.05% TWEEN 40-0.5% CHLORAMPHENICOL-250mg/L
         Stock inoculum was prepared. Anti-fungal stock solutions of drugs were
prepared and stored at - 700C. Micro titre plates with 96 wells were incubated
for 4 days at 32oC. Growth and sterility control wells were included in each test.
MIC values to anti-fungal drugs - Fluconazole, Miconazole, Clotrimazole,
Sertaconazole and lulliconazole were obtained as MIC 50 and MIC90 (where 50%
and 90% inhibition of growth was seen).

Molecular study-

DNA extraction was done by phenol: chloroform method and stored at -20O C
until used as a template for amplification.

DNA amplification

PAN FUNGAL PCR (TABLE 1)

Pan fungal PCR was done on 20 samples using ITS -1 and ITS-4 primers

ITS-1 5΄ TCCGTAGGTGAACCTGAGG3΄

ITS-4 5 ΄ TCCTCCGCTTATTGATATGC3΄

MALASSEZIA SPECIFIC NESTED PRIMER (TABLE 2)

Pan fungal PCR positive samples were subjected to species specific nested
PCR using Malassezia specific primers

ITSIF-N 5΄GGATCATTAGTGATTGCCTTTATA 3΄ and

ITS4-R 5΄ TCCTCCGCTTATTGATATG 3΄

Sequencing -Sanger’s sequencing was done

Blasting-NCBI blast was done

RESULT
Of the 88 samples collected 78 showed meat ball and spaghetti appearance on microscopic examination (85%)
65 samples grew on MDA. Some samples even grew more than one species of Malassezia.
50 samples grew on SDA with olive oil overlay.
Growth on Modified Dixons agar was faster than SDA with Olive Oil overlay.
Gram stain performed showed-spherical/oval/sympodial buddings
All samples were catalase and Oxidase positive.
Predominant age group 20-30 yrs.
Sex predominance showed little female predominance.
Commonest site -Scalp, Neck and Shoulder, Hip and inner thigh
RPMI1640 with added supplements were standardised
The AFST reports are awaited
Molecular Methods were standardised and DNA Extracted and stored for Amplification.
Waiting for Sequence Reports, Blasting and Speciation.

CONCLUSION

    The commonest species causing infections was MALASSEZIA sympodialis,M.furfur,M.globosa and M.obtusa.Clotrimazole showed the lowest MIC values followed by Lulliconazole, Miconazole and Sertaconazole. Fluconazole showed comparatively higher MIC values.
     Speciation and anti-fungal susceptibility is very important to prevent the chronicity in treatment and have a good patient compliance.

3 / Molecular identification and speciation of Malassezia and its susceptibility pattern