Shivaprakash M Rudramurthy Additional Professor, Department of Medical Microbiology, PGIMER, Chandigarh

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1 Shivaprakash M Rudramurthy Additional Professor, Department of Medical Microbiology, PGIMER, Chandigarh

2 Commensal Unipolar budding Lipophilic Resides in area rich in sebaceous gland Early difficulty in the isolation in the laboratory held back research Introduction of molecular methods has accelerated work on the genus in recent years

3 Topographic diversity of fungal and bacterial communities in human skin Findley et al. Nature. 2013

4 SD/D PV Folliculitis SD Psoriasis AD

5 Association between Malassezia and dermatoses Malassezia Dermatoses Presence of organism on the affected skin Clear therapeutic response to antifungals Reduction in Malassezia load after treatment

6 Several complex mechanisms underlying the dermatoses associated with Malassezia Host genetic factor Immune response to free fatty acids/proteins/polysaccharides from Malassezia Innate differences in stratum corneum barrier function Skin permeability Hormonal Malassezia Host genetic factor Environment Disease aggravation PV- most common in summer SD/D- most common during winter Enviornment

7 Problems associated with Malassezia Absolute requirement of lipid supplements Defect in the synthesis of myristic acid- serves as the precursor of long-chained fatty acids Absence of fatty acid synthase gene Variable survival rate Overgrowth of fast growing species In culture based methods M. restricta and M. obtusa grow relatively slowly, and M. furfur and M. sympodialis grow rapidly. A slow-growing strain may be missed.

8 Problems associated with Malassezia Results are not reproducible The recovery is dependent on sampling methods and isolation media. Isolation of a pure, single colony of a Malassezia species is sometimes difficult. Difficulty in storage & Loosing viability

9 Geographical variations in the isolation of Malassezia species North Europe- M. sympodialis- HC/AD/SD South Europe- M. globosa HC/PV/SD. M. obtusa is frequently isolated AD/HC North, but no M. obtusa isolation has been reported in the South Europe. In Japan- HC/AD Description of the new species M. dermatis, M. japonica and M. nana. Boekhout et al, Malassezia and the skin, 2012

10 Existence of pathogenic species /strains within species Healthy Vs diseased skin isolates Effect of environmental factors on Malassezia-associated diseases Quantitative data on the distribution of the newly described species on the human skin is still awaited.

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12 Pityriasis versicolor (PV) most common chronic superficial infection of the stratum corneum Reported in 40-60% tropical population

13 Pityriasis versicolor Predisposing factors- largely unknown Genetic susceptibility plasma cortisol level Temp. & humidity Malnutrition Hyperhidrosis

14 Distribution of Malassezia species in patients with pityriasis versicolor

15 Malassezia spp. Distribution in lesional area (88%) M.slooffiae M.sympodialis (2.3%) (4.5%) M.furfur+ M.globosa (15.9%) M.globosa (27.3%) Malassezia spp. distribution In non-lesional area (40%) M.furfur+ M.slooffiae M.globosa (5%) (5%) M.globosa (5%) M.furfur (50%) M.furfur (85%) Malassezia spp. distribution in healthy individuals (60%) M.sympodiali s (10%) M.furfur + M.globosa (16.7%) M.furfur (73.3%)

16 Malassezia colony counts Mean colony count from PV patients Lesional area 18.6 cfu and 8 confluent p<0.05 Non- lesional area 7.9 cfu and 1 confluent Mean colony count from healthy individuals Forehead 10 cfu Cheek 10 cfu Chest 12 cfu

17 Relapse is very common, particularly in patients living in the tropics Prophylactic treatment with propylene glycol in water - prevent relapses Faergemann J et al, (1980) Acta Derm Venereol Others- tried periodically applied ketoconazole shampoo There is no clinical trial data

18 Higher Malassezia density was found in lesional area compared to non-lesional area of PV patients and in healthy individuals (p<0.05). M. furfur was the most prevalent species isolated from both patients and control Significant- isolation of M. globosa from the lesional area (27.3%) compared to non-lesional area (5%) (p<0.05). Possible role of M. globosa in causing PV

19 Discomfort through irritation Can lead to low self esteem & a negative social image For SD alone, the health care direct & indirect costs exceeded $1.4 billion in the United States in 2004 Naldi L, NEJM, 2009

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21 Distribution of Malassezia species in patients with seborrhoeic dermatitis

22 Study population: Punjab- 50 SD/D patients + 10 healthy Coastal karnataka- 50 SD/D patients + 10 healthy Shivaprakash et al, IJMR, 2014

23 Malassezia spp. distribution in SD/D patients & healthy individuals Study group No of Malassezia isolated (%) North India 37 (74%) South India 47 (94%) Healthy Individual 6 (30%) North India M. slooffiae (5.4%) M. restricta (2.2%) M. furfur (8.1%) South India M. globosa (10.6%) M. sympodialis (8.1%) M. restricta (40.5%) M. globosa (37.9%) M. furfur (34%) M. furfur + M. restricta (25.5%) M. globosa + M. restricta (27.7%)

24 AFLP_6-FAM AFLP_6-FAM A10_B02_ _u. A6_F01_ _un. A19_C03_ _u. A12_D02_ _u. A11_C02_ _u. A55_G07_ _. A51_C07_ _u. A57_A08_ _u. A30_F04_ _u. A56_H07_ _u. Type II A8_H01_ _un. A75_C10_ _u. A3_C01_ _un. A4_D01_ _un. A16_H02_ _u. A61_E08_ _u. A48_H06_ _u. A14_F02_ _u. A65_A09_ _u. A78_F10_ _u. A50_B07_ _u. A70_F09_ _u. A39_G05_ _. A47_G06_ _. A32_H04_ _u. A33_A05_ _u. A68_D09_ _u. A72_H09_ _u. A76_D10_ _u. A40_H05_ _u. A67_C09_ _u. A54_F07_ _u. A71_G09_ _. A59_C08_ _u. Type I M. restricta A46_F06_ _u. A36_D05_ _u. A37_E05_ _u. A43_C06_ _u. A64_H08_ _u. A77_E10_ _u. A38_F05_ _u. A52_D07_ _u. A35_C05_ _u. A44_D06_ _u. A28_D04_ _u. A28_D04_ _u. A42_B06_ _u. A42_B06_ _u. A49_A07_ _u. A26_B04_ _u. A73_A10_ _u. A25_A04_ _u. A13_E02_ _u. A5_E01_ _un. A18_B03_ _u. A58_B08_ _u. A69_E09_ _u. A15_G02_ _. A20_D03_ _u. A60_D08_ _u. M. arunalokei A7_G01_ _un. A17_A03_ _u. A79_G10_ _. A21_E03_ _u. A29_E04_ _u. A41_A06_ _u. A53_E07_ _u. A9_A02_ _un. A23_G03_ _. A2_B01_ _un. M. furfur A22_F03_ _u. A24_H03_ _u. A80_H10_ _u. Type III A34_B05_ _u.

25 On the basis micromorphological features phenotypic characteristics PCR-RFLP of ITS2 region (Bgl1) Sequence divergence observed D1/D2 region of 26S rdna ITS-5.8S region of rdna IGS1region rdna FAFLP Microsatellite fingerprint typing MALDI-TOF MS data We proposed a new species in the Malassezia genus and designated it as M. arunalokei sp. nov.

26 Comparison of phospholipase between SD and healthy control isolates ** * * β-endorphin exposure modifies the in vitro phospholipase activity in Malassezia species isolated from SD lesional skin.

27 M. globosa and M. restricta are predominant species causing dandruff in Indian population High density of these species on the scalp correlates with severity of dandruff Description of new species M. arunalokei (15th Malassezia spp.) β-endorphin exposure modifies the in vitro phospholipase activity in Malassezia species isolated from SD lesional skin

28 Psoriasis is a disease characterized by hyperproliferation and hyperkeratinization of the epidermis Microbial environmental factors Immunological genetic component The epidemiological data on the distribution Malassezia spp. in psoriasis skin are sparse & contradictory

29 Distribution of Malassezia species in patients with psoriasis

30 The spectrum of Malassezia spp. isolated from (a) lesional, (b) non-lesional site of psoriasis patients and (c) healthy individual. Distribution of Malassezia species on different sites of psoriatic lesions. Shivaprakash et al, Mycoses, 2014

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32 Malassezia colony counts Mean colony count from psoriasis patients Lesional area 17 cfu Non- lesional area 16 cfu Mean colony count from healthy individuals Forehead Cheek Chest 10 cfu 10 cfu 12 cfu

33 Mean colony count from lesional and non-lesional area of psoriasis patient skin No of Site of sample Mean colony count Mean colony count patients collection from lesional area from non-lesional area 11 Chest Scalp * 8 Arms Abdomen Back Neck Other sites P value

34 No consistent pattern was observed between frequency and density of Malassezia species with psoriasis lesions as compared to healthy subjects. Malassezia may be the important etiological agent in exacerbation of scalp psoriasis. Malassezia species probably may not have significant role in exacerbating psoriasis lesions.

35 Burden of Malassezia associated dermatoses in India Dermat Prevalen Studies ological ce/ conducted problem incidence Pityriasi s versicol or 40-60% Isolation Predominant rate Malassezia spp. Comments Chandigarh, 88% Allahabad, Chennai, Patiala M. furfur & M. globosa prevalence due to hot and humid climate. Colony count higher in lesional area compared to non lesional area Psoriasis 0.44% to 2.8% all patients with skin diseases Chandigarh 68% M. furfur No significant difference in burden of Malassezia in between lesional and non lesional area, Except for scalp psoriasis Sebo.de varies rmatitis/ between dandruff 30-95% Chandigarh 74% M. restricta and M. globosa Colony count higher in lesional area compared to non lesional area Healthy control Chandigarh 60% M. furfur

36 Table 1. Our experience, PGIMER, Chandigarh, India Dermatological problem Isolation rate Lesional area Non-lesional area Pityriasis versicolor 88% M. furfur- 50% M. globosa- 27% M. furfur+ M. globosa- 16% M. furfur- 85% M. globosa- 5% M. furfur+ M. globosa- 5% Psoriasis 68% M. furfur- 71%, M. japonica- 12%, M. globosa- 9% M. furfur- 71%, M. slooffiae- 14% M. globosa- 9% Sebo.dermatitis/ dandruff 74% Healthy control 60% M. restricta- 41%, M. globosa- 38%, -Isolation rate in agreement M. furfur- 8%, M. sympodialis- 8% with collective data from different epidemiological studies M. furfur- 73% M. furfur+ M. globosa- 17% Table 2. Studies on pityriasis versicolor, India Siddharth et al, Alhabad Kindo et al, Chennai Kaur et al, Patiala Int J Derm IJMM IJMM 70 scrapings- 48 growth 427 scrapings 250 growth 58 scrapings- 54 growth M. sympodialis- 58% M. globosa- 40% M. restricta - 2% M. globosa 136, M. furfur 74, M. globosa + M. sympodalis 12 M. globosa + M. furfur 11 Unidentified spp. 8 M. globosa- 51% M. sympodialis- 31% M. furfur- 18% Identification by- conventional methods

37 InflammatoryPredicted postulates Sensitization to cross-reactive allergens produced by Malassezia yeasts Damaging the epidermal barrier function through the production of lipases and phospholipases inflammatory response Local immune response through local production of cytokines

38 Molecular Epidemiology PCR-based methods- RAPD, PCR-typing, RFLP, AFLP emerged to answer different research questions In future expected- Development of diagnostic (micro)array platforms A promising method- MLST Unfortunately, has not yet been used in epidemiological studies of Malassezia-related studies Advantage Results are reliable and reproducible Storing of the MLST data in a central depository

39 Epidemiological Investigations by RealReal-time PCR Identification and quantification- Malassezia DNA from skin specimens Without the need to culture isolates Quantified using Taq-Man probes Sampling is performed using tape or a swab, and fungal DNA is subsequently extracted directly from the collected samples and analyzed by PCR

40 Epidemiological Investigations by RealReal-time PCR Limitations Unable to utilize isolates for pathogenesis studies Production of lipases Production of Indole Melanin synthesis Unable to utilize isolates for AST and drug resistance studies Quantifies DNA from non-viable and metabolically inactive cells also Unable to quantify the Malassezia which resides in the inflandibular region-the normal habitat

41 Malassezia Fungemia and Invasive Infections Malassezia species - rare causes of invasive infections Critically ill low-birth-weight infants Immunocompromised children and adults Many routine blood culture systems do not effectively support its growth in vitro Incidence will most likely be even higher as current clinical data suggest Molecular methods offer some hope for improved detection

42 Malassezia Fungemia and Invasive Infections The clinical spectrum- asymptoma c infec on lifethreatening sepsis and disseminated disease Risk factors Intravascular catheters Administration of lipid supplemented parenteral nutrition

43 Antifungal susceptibility testing Lack of standardization for Malassezia susceptibility testing restricts associations of in vitro with in vivo responses to antifungals Systematic studies correlating clinical outcome with susceptibility results may give definitive conclusions Appropriate validation (standardization) is required before adopting a susceptibility assay intended for patient care and welfare Standardized susceptibility testing - co-evaluated with pharmacodynamic and pharmacokinetic studies in natural systems (human stratum corneum)

44 These controversies demonstrate the multifaceted interactions of Malassezia yeasts with the skin Diversity of the clinical presentations of Malasseziaassociated diseases does not allow the formation of solid pathogenetic pathways In future- fascinating research will highlight the role of this yeast on skin physiology