DANDRUFF AETIOLOGY AND THE EFFECTS OF EDIBLE LIPIDS ON THE GROWTH OF ISOLATES

Abstract

Background: Dandruff is caused by Malassezia species, a lipophilic fungus. Information on the aetiology of the fungi associated with this condition and profile of affected persons are sparse in our locality. This prospective study was designed to determine the aetiology of dandruff causing fungi and to assess the effects of some edible lipids on the growth of predominant isolates in our locality. Methods: The participants enrolled for the study were 245 in number. Those with dandruff-like lesions were 145 and those without lesions (controls) were 100. Ethical approval was obtained. Questionnaires were administered for biodata. Scalp scrapings were obtained from subjects visiting hair and beauty salons into sterile paper envelopes and transported to the Microbiology Laboratory, UCTH, for analysis. Samples were subjected to culture, microscopy and physiological tests. Growth response of Malassezia species to lipids was assessed using common edible oils including groundnut oil. Results: Out of the 145 subjects with dandruff-like lesions, 90.3% were positive for different Malassezia species. The most prevalent Malassezia species among subjects with dandruff lesions was M. furfur (70.2%) while M. globosa (51.9%) was the most prevalent among the controls. Malassezia pachydermatis was the least prevalent species among the two groups. M. furfur grew exceptionally on SDA overlaid with groundnut oil. Conclusion: Groundnut oil could be used as an alternative for the cultivation of M. furfur especially in a resource poor setting like ours and should not be used in the preparation of hair ointment or creams.

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Ogba et al. European Journal of Pharmaceutical and Medical Research
71
DANDRUFF AETIOLOGY AND THE EFFECTS OF EDIBLE LIPIDS ON THE
GROWTH OF ISOLATES
Nengimoyo Biobelemonye1, Ofonime M. Ogba1* and Lydia N. Abia-Bassey1
Department of Medical Laboratory Science, Faculty of Allied Medical Sciences, University of Calabar, Nigeria.
Article Received on 05/07/2016 Article Revised on 25/07/2016 Article Accepted on 15/08/2016
INTRODUCTION
Dandruff, medically known as Pityriasis capitis is a
condition, characterized by small white flakes of skin
scales that separate and fall from the scalp. It is a
condition that causes social or self esteem problems to
those affected.[1] Symptoms include: itching, reddening
and flaking in areas with a rich supply of sebum
production. Lesions are red and covered with greasy
scales. There are more chances of hair lose on scalp with
dandruff than non-dandruff scalps which shows the
relationship with Malassezia infection.[1]
Dandruff affects more than 50% of adult population and
represents 25% of all scalp disorders.[2-4] A USA survey
reported that about 50 million people suffer dandruff
infections yearly and about $300 million is spent
annually on different treatment options.[5-6]
Malassezia is the only known fungi implicated in
Dandruff. The different species include; M. furfur, M.
globosa, M. pachydermatis, M. sympodialis, M. obtuse,
M. slooffiae and M. restricta. However, the dominant
species on human scalp are Malassezia furfur,
Malassezia globosa and Malassezia restricta.[2]
Malassezia are lipophilic fungi.[7-8] People with over
active sebaceous glands suffer from repeated attack of
dandruff because of their oily scalp.[9] Malassezia
proliferates more and deregulates keratinization.[10] The
keratinized cells clump together resulting in large flakes
of scales. The scales can be washed away with agents
that lyse keratinized cells, example, selsun blue and
salicylic acid.[10]
There is a suggestion that the presence of androgens in
males play a role in their higher susceptibiity to dandruff
than females. People who are bald do not suffer this
condition because the hair follicle is necessary for fungal
colonization.[11]
Malassezia colonies are cream colored, yellow to brown,
orange or beige depending on the species. They are
raised and smooth initially but become dry and wrinkled
with age. This organism is a fast grower and mature in 5
days at 30-37°C. Growth is poor at 25°C incubation.[12]
Dandruff predisposing factors in immunocompetent and
immune-compromised individuals are multifactoral, they
may be Intrinsic, Extrinsic or Pathogen related.[13]
Intrinsic factors include; Genetic predisposition,
emotional component, increase in the alkalinity of the
skin and neurological diseases.[14] Extrinsic factors
SJIF Impact Factor 3.628
Research Article
ISSN 3294-3211
EJPMR
EUROPEAN JOURNAL OF PHARMACEUTICAL
AND MEDICAL RESEARCH
www.ejpmr.com
ejpmr, 2016,3(9), 71-76.
*Correspondence for Author: Dr. Ofonime M. Ogba
Department of Medical Laboratory Science, Faculty of Allied Medical Sciences, University of Calabar, Nigeria.
ABSTRACT
Background: Dandruff is caused by Malassezia species, a lipophilic fungus. Information on the aetiology of the
fungi associated with this condition and profile of affected persons are sparse in our locality. This prospective study
was designed to determine the aetiology of dandruff causing fungi and to assess the effects of some edible lipids on
the growth of predominant isolates in our locality. Methods: The participants enrolled for the study were 245 in
number. Those with dandruff-like lesions were 145 and those without lesions (controls) were 100. Ethical approval
was obtained. Questionnaires were administered for biodata. Scalp scrapings were obtained from subjects visiting
hair and beauty salons into sterile paper envelopes and transported to the Microbiology Laboratory, UCTH, for
analysis. Samples were subjected to culture, microscopy and physiological tests. Growth response of Malassezia
species to lipids was assessed using common edible oils including groundnut oil. Results: Out of the 145 subjects
with dandruff-like lesions, 90.3% were positive for different Malassezia species. The most prevalent Malassezia
species among subjects with dandruff lesions was M. furfur (70.2%) while M. globosa (51.9%) was the most
prevalent among the controls. Malassezia pachydermatis was the least prevalent species among the two groups. M.
furfur grew exceptionally on SDA overlaid with groundnut oil. Conclusion: Groundnut oil could be used as an
alternative for the cultivation of M. furfur especially in a resource poor setting like ours and should not be used in
the preparation of hair ointment or creams.
KEYWORDS: Malassezia pachydermatis, M. globosa.

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Ogba et al. European Journal of Pharmaceutical and Medical Research
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include; too much application of oils on hair with scalp,
use of wrong hair conditioners, not rinsing hair properly
after application of conditioners and poor hair
hygiene.[15] Pathogen factor include; lipase production,
metabolism of triglycerides and oleic acid synthesis.[14]
There is paucity of information on pityriasis capitis
aetiology, and the profile of persons affected in our
locality. This study was carried out to determine the
aetiology and types of dandruff-like causing fungi among
individuals visiting hair salons in Calabar and the
prevention and management of this condition.
MATERIALS AND METHODS
Study area
The study area was Calabar, Nigeria, the capital of Cross
River State. She is located at (4o57N, 8o19E). The state
falls within the Tropical Zone. There are two seasons, the
rainy season from May-October with a mean annual
rainfall of 243-336mm and dry season from November-
April. This period may change from year to year. It has a
daily temperature which varies between 27oC-31oC with
small annual range of 4oC and 5oC. Relative Humidity is
high for the rainy season (80-90%) and low in the dry
season (45-50%).[16]
Subject selection/Ethical approval
Subjects for this study were enrolled after obtaining due
approval from the Ethical Research committee. Informed
consents were also obtained from the subjects. A pre-
designed protocol was administered to all the subjects for
information on biodata, treatment options and presenting
complaints.
Sample collection
Convenient samples were obtained from subjects of both
gender visiting hair salons and barber shops located in
the two Local Government Areas in the State capital,
between December 2012 and May 2013. Control subjects
were male and female students who reside in the
University of Calabar hostels. Human scalp scrapings
were obtained from 245 subjects; 145 subjects with
dandruff-like lesions and 100 with no sign of dandruff-
like lesions (controls) into sterile paper envelops.
Samples were transported to the Microbiology
Laboratory, University of Calabar Teaching Hospital for
analysis.
Microscopic examination
Scalp scrapings were examined as wet mounts in 10%
KOH for thick-walled round, yeast-like cells alongside
short angular hyphae which are characteristic features of
Malassezia species.[17]
Culture
Samples were cultured on Sabouraud Dextrose Agar
containing 0.5mg chloramphenicol overlaid with sterile
olive oil.[18] Plates were incubated for 7 days at a
temperature of 370C. The agar plates were examined
every other day for fungal growth.[18]
Identification of isolates
Pure cultures of every isolate were prepared before
performing any physiological test. Isolates were
identified by gross and microscopic morphology
alongside physiological tests. Microscopic examination
of isolates was in Lactophenol cotton blue stain.
Identification and speciation of isolates was carried out
using the following test; catalase test, Lipid test and
Tween assimilation test.[19]
Physiological tests
Catalase Test
Catalase production was used to differentiate M. restricta
from M. obtuse, M. globosa and M. furfur. All
Malassezia species produce catalase except M. restricta.
Malassezia pachydermatis have variable reaction.[20-21]
Lipid test
The yeast isolates from pure cultures were inoculated on
SDA containing 0.5mg chloramphenicol in duplicates.
One was overlaid with sterile olive oil and the other was
not. The plates were incubated for 7 days at 37oC.
Growth rate was observed to determine lipid dependent
and non-lipid dependent species of Malassezia.
Tween Assimilation Test
This test was performed to differentiate Malassezia
furfur from M.globusa and M. obtuse. The test was
evaluated according to Guillot et al.[22] methods. A 24hr
culture of the isolates was used to prepare the inoculum.
The organisms suspension was constituted in 5ml of
sterile normal saline. The turbidity was adjusted to 0.5
McFarland standard which is an equivalent of 1 x 106 to
5x106 CFU/ml. One ml of the McFarland adjusted
culture was inoculated on Sabouraud Dextrose Agar
plate. After the surface has dried, four holes were made
with sterile 2mm diameter punch and filled with 5μl of
Tween 20, 40, 60 and 80 (Shreeji Pharma International,
India) respectively. The plates were incubated for 7 days
at 37°C. Tweens utilization was assessed by the degree of
growth and/or precipitation of the lipid loving yeasts
around the wells and their rate of growth.[21-22] Plates
with visible growth were incubated at 40°C for 48 hours,
only M. furfur can survive this temperature.
Urease test
Urease test was positive only for M. furfur and M.
pachydermatis.
Effect of selected oil on the growth of M. furfur
Commonly available edible oils were selected to assess
the growth rate of M. furfur when overlaid with them.
The oils were palm oil, margarine, coconut oil,
groundnut oil, olive oil, castor oil and palm kernel oil.
All the oils were procured in the market and sterilized at
160oC in hot air oven for 60 minutes. Sterile swab sticks
were dipped into sterile Tween 80 and used to pick pure
distinct colonies of the yeast to suspend in 3ml of sterile
normal saline and vortex. The suspension turbidity was
adjusted to 0.5 McFarland standards by visual inspection.

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Ogba et al. European Journal of Pharmaceutical and Medical Research
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Sabouraud dextrose agar plates prepared in duplicates for
each isolate were overlaid with 2ml of the various oils.
All plates were inoculated with 1ml of 0.5 McFarland
adjusted culture suspension by the use of an automatic
pipette. All cultures were incubated for 48hrs at 37oC or
until good growth was seen in the control. Olive oil
plates were used as control because it is the standard
recommended oil for the growth of M. furfur. Growth on
the plates were compared to that of the control (olive oil
plate) and scored by visual inspection. They were graded
as shown follows;
++++ - Excellent growth
+++ - growth same as control
++ - Fair growth (less than that of control)
+ - Poor growth
RESULTS
Out of the 245 subjects enrolled for the study,
185/245(75.5%) were positive for Malassesia infection.
The prevalence rate was 90.3% (131/145) among
subjects with dandruff-like lesions and 54.0% (54/100)
among those without dandruff lesion (Table 1).
TABLE 1: Prevalence of Malassezia isolates among subjects.
Status of Malassezia
infection
No. (%) of Subjects with
Dandruff-like lesions
(n=145)
No. (%) of Subjects
without dandruff-like
lesion (n=100)
Total
(n=245)
Malassezia positive
131(90.3)
54(54)
185(75.5)
Malassezia negative
14(9.7)
46(46)
60(24.5)
Figure 1 shows the distribution of Malassezia species
among subjects. Malassezia furfur 92(70.2%) and M.
globosaI 31(23.7%) were more predominant species
among subjects with dandruff-like lesions while M.
globosa 28(51.9%) and M.restricta 14(25.9%) were
more predominant among subjects without lesions.
Fig 1 Distribution of Malassezia species among
subjects
Plate 1 shows a photograph of a human scalp in health
and with dandruff. Plate A shows a healthy scalp
while B shows a scalp with Pityriasis capitis lesions
alongside white flaky scales on the hair.
B
A
PLATE 1 Photograph of a healthy and dandruff scalp
Table 2 shows the distribution of Malassezia infection
among subjects by age. The highest Malassezia infection
rate 51.9% (96/109) was seen in age 25-34 years
followed by 27.6%(51/63) among age 15-24 years while
the least 0.5%(1/5) was seen among age 55-65 years.

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Ogba et al. European Journal of Pharmaceutical and Medical Research
74
TABLE 2: Distribution of Malassezia species among subjects by age
Figure 2 shows the symptoms and complaints from
subjects with Pityriasis capitis. The commonest
presenting symptoms were itching (28.9%), flaking or
scaling (23.5%), social/emotional discomfort (16.6%)
and alopecia (15.2%). Other less frequent complaints
were wound on the scalp (7.6%), headache (2.1%) and
erythema (6.2%).
Fig 2 Symptoms associated with Pityriasis capitis
Table 3 shows the characterization of Malassezia species
by physiological and biochemical tests. The tests carried
out were; Tween assimilation test, Catalase test, Urease
test, Growth at 40oC and Growth with or without the
addition of olive oil.
Table 4 shows the growth assessment of M. furfur using
seven edible lipids in the locality. These include:
groundnut oil, margarine, castor oil, palm oil, palm
kernel and coconut oil. Olive oil was used as a control.
Among the seven lipids, M. furfur grew exceptionally on
SDA overlaid with groundnut oil. Good growth was also
observed on SDA overlaid with coconut oil and
margarine but growth was poor on SDA with palm
kernel oil and palm oil.
Table 3 Characterization of Malassezia species.
(Soo-Jung et al.[23]
KEY
+ = presence of growth
- = absence of growth
TABLE 4: Growth assessment of Malassezia furfur on
Sabouraud Agar overlaid with edible oils in the
locality.
Edible oils
Growth rate
Groundnut oil
++++
Coconut oil
+++
Olive oil
+++
Margarine
+++
Castor oil
+++
Palm oil
+
Palm Kernel oil
+
++++ = Excellent Growth, +++ = Good Growth, ++ =
Fair Growth, + = Poor Growth.
DISCUSSION AND CONCLUSION
Discussion
Information on the aetiology of dandruff causing fungi
and the profile of affected persons are sparse in Calabar,
Nigeria. This study has provided useful information on
these areas.
Malassezia was isolated from most (90.3%) of the
subjects with dandruff-like lesions however, 54% of the
control subjects were colonized. This suggests that
Malassezia may be a normal flora on healthy scalp. On
the other hand, Malassezia furfur, the most predominant
species among the test subjects may have caused
dandruff-like lesions and its associated symptoms.
Age (Years)
Total No.
examined
No. (%) positive for
Malassezia
15-24
63
51(27.6)
25-34
109
96(51.9)
35-44
36
26(14.1)
45-54
32
11(5.9)
55-65
Total
5
245
1(0.5)
185
Malassezia Species
Urease
test
No Lipid
supplement
Lipids
supplement
Tween
20,40,80
Catalase
reaction
Growth
at 40oc
M. furfur
+
-
+
+
+
+
M. globosa
-
-
+
-
+
-
M. restricta
-
-
+
-
-
-
M. pachydermatis
+
+
-
+
+
-

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Ogba et al. European Journal of Pharmaceutical and Medical Research
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Although sebum production was not investigated in our
study, the highest prevalence of dandruff-like lesions
was seen in subjects aged 25-34 years which correspond
with the age of the peak production of sebum which is
usually at post-puberty.[23-24] An evaluation of sebum
production may be needed to assess its role in the
production of dandruff and growth of Malassezia.
Subjects with dandruff-like lesions presented more than
one symptom and complaints. The commonest
complaint/symptom were itching, scaling and loss of
hair, this agrees with the reports of Nematian et al.[1] who
reported flaking and hair loss as the predominant
symptom of dandruff.
It is interesting to note that the species M. pachydermatis
also isolated in our study are known to be found in pet
animals which suggest a zoonotic transmission. However
data on pet reference was not obtained from our subjects.
CONCLUSION
Malassezia furfur and M. globosa could jointly cause
dandruff. Groundnut oil enhances the growth of M.
furfur and could be used as an alternative for the
cultivation of M. furfur especially in resource poor
settings like ours. On the other hand it should not be used
in the preparation of hair ointment or creams. Further
investigations should be done to evaluate sebum
production and its association with Malassezia and
dandruff formation.
ACKNOWLEGEMENT
We are grateful to the management and staff of the
selected beauty salons in Calabar for the use of their
customers as subjects, to UCTH for the use of their
facilities and University of Calabar for the enabling
environment for research.
Competing Interest: None.
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