Mycopathologia (2011) 171:235250

DOI 10.1007/s11046-010-9373-7

A Review of the Ultrastructural Features of Superficial

Candidiasis
J. A. M. S. Jayatilake

Received: 1 February 2010 / Accepted: 30 September 2010 / Published online: 22 October 2010
Springer Science+Business Media B.V. 2010

Abstract Commensal yeast Candida causes opportunistic infections ranging from superficial lesions to
disseminated mycoses in compromised patients.
Superficial candidiasis, the commonest form of
candidal infections, primarily affects the mucosa
and the skin where Candida lives as a commensal.
Conversion of candidal commensalism into opportunism at the fungalepithelial interface is still illdefined. Nevertheless, fungal virulence mechanisms
such as adhesion to epithelia, morphogenesis, production of secretory hydrolytic enzymes, and phenotypic switching are thought to contribute in the
process of pathogenesis. On the other hand, host
responses in terms of immunity and local epithelial
responses are actively involved in resisting the fungal
challenge at the advancing front of the infection.
Ultrastructural investigations using electron microscopy along with immunohistochemistry, cytochemistry, etc. have helped better viewing of Candidahost
interactions. Thus, studies on the ultrastructure of
superficial candidiasis have revealed a number of
fungal behaviors and associated host responses such
as adhesion, morphogenesis (hyphae and appresoria
formation),
thigmotropism,
production
and

distribution of extracellular enzymes, phagocytosis,

and epithelial changes. The purpose of this review is
to sum up most of the ultrastructural findings of
Candidahost interactions and to delineate the
important pathological processes underlying superficial candidiasis.
Keywords Candidiasis
Candida species

Cytochemistry
Electron microscopy

Histopathology
Immunohistochemistry
Abbreviations
CAM
Chick chorio-allantoic membrane
HIV
Human immunodeficiency virus
ICMS Intracytoplasmic membrane structures
ICMT Intracytoplasmic membrane tubules
PL
Phospholipase
PLB
Phospholipase B
PMNL Polymorphoneuclear leukocytes
SAP
Secretory aspartyl proteinase
SEM
Scanning electron microscope
TEM
Transmission electron microscope

Introduction
J. A. M. S. Jayatilake (&)
Division of Microbiology, Department of Oral Medicine
and Periodontology, Faculty of Dental Sciences,
University of Peradeniya, Peradeniya, Sri Lanka
e-mail: sumedhaj@pdn.ac.lk

Candida species are common commensals of

humans. However, they incite opportunistic infections ranging from less-severe superficial lesions to

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life-threatening disseminated mycoses in compromised patients. Superficial candidiasis commonly

affects the mucosa and the skin. Moreover, skin
appendages such as nails and hair could also be
affected by candidal infections [1]. Disseminated
candidiasis is characterized by the spread of fungi
through the tissues and blood circulation. Incidence
of either types of candidiasis has shown a virtual
surge due to the increased number of compromised
patients with HIV infection, organ transplants, and
endocrine disorders such as hypothyroidism and
diabetes mellitus [2, 3]. In addition, widely used
modern therapeutics including broad-spectrum antibiotics, steroids, and cytotoxics have also contributed
to a rise in the incidence of candidiasis. Superficial
candidiasis is the commonest form of candidiasis
primarily affecting the mucosa of the human oral
cavity and the vagina where Candida inhabits as a
commensal organism [4]. Oral candidiasis is considered as one of the premonitory conditions of HIV
infection at present. On the other hand, it is reported
that 75% of female population experience vaginal
candidiasis at least once in their life span [5].
Putative virulence attributes that help conversion
of Candida from an innocuous commensal organism
to an opportunistic pathogen are adhesion, hyphal
invasion, production of extracellular enzymes such as
phospholipases (PL) and secretory aspartyl proteinases (SAP), and phenotypic switching [4]. Invasion of
fungal elements assisted by the above virulence
attributes through the epithelial barrier is pathognomonic in superficial candidiasis. Interestingly, ultrastructural studies using a variety of tissue specimens
derived from clinical and experimental candidiasis
have identified a number of fungalhost interactions
at the invasive interface between Candida and the
epithelium. This review focuses on the ultrastructural
features of the fungalhost interactions observed in
superficial candidiasis with a particular reference to
the contribution of Candida virulence in the evasion
of host immunity leading to invasive pathogenesis.

Methods
State of knowledge in the field of ultrastructural
pathology of superficial candidiasis on the basis of
selective review of English literature in various
sources was critically analyzed. These literature

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searches were primarily based on Medline literature

that included key words such as Candida, ultrastructure, invasion, and epithelium followed by subsequent suitable subheadings. Appropriate medical and
dental text books on superficial candidiasis were also
referenced. Additionally, relevant literature on electron microscopic investigations regarding fungi was
reviewed. While the search spanned the years from
1968 to the present, recent literature was prioritized.
Selected information was extrapolated based on the
clinical and scientific relevance. Thus, accumulating
evidence suggests that multiple fungalhost interactions occur in superficial candidiasis.

General Morphology of Candida

Vegetative cells of Candida species are globose or
ellipsoidal and produce daughter cells by multilateral
budding [6]. However, different Candida species
exhibit unified shapes and sizes of vegetative blastospores (yeast cells) of 25 lm when observed
under the light microscope. For example, C. albicans
blastospores are oval and spherical, whereas C. krusei
are elongated with a characteristic long rice-grain
appearance [7]. Except C. glabrata, other Candida
species form filamentous structures known as true
hyphae or pseudohyphae in favorable growth conditions, whereas C. albicans and C. dubliniensis share
the ability to form both true hyphae and pseudohyphae. Pseudohyphae result from buds and are essentially elongated blastospores unseparated in the
process of budding. In contrast, true hyphae have
almost perfectly cylindrical cell wall, since they are
formed by the outgrowths of blastospores (germ
tubes) that elongate apically.
Candida species also produce chlamydospores,
thick-walled refractile spheres attached to pseudohyphae by an elongated cell or a suspensor cell.
Chlamydospores differ from blastospores by larger
size and spherical shape and refractile appearance
under the light microscope. Both C. albicans and C.
dubliniensis produce chlamydospores in nutrientdepleted media [8, 9]. In different favorable environments, C. albicans transforms into all these morphogenic states (blastospore, pseudohypha, true-hypha,
and chlamydospore), and therefore, sometimes have
been known as polymorphic [10, 11].

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Laboratory Techniques
Conventional light microscopy shows that candidal
blastospores as well as hyphae penetrate into the
epithelium during superficial candidiasis. However,
fine features of these interactions are visualized using
transmission and scanning electron microscope.
Transmission (TEM) and scanning electron microscopy (SEM) provide bi- and tri-dimensional views of
tissue specimens affected by the fungi, respectively.
However, ultrastructural investigations of Candida
are hampered particularly by the technical difficulties
in processing yeasts for electron microscopy [12, 13].
Precise imaging of yeasts depends on the specific
fixatives, and staining chemicals used in the preparation of specimens for microscopy. Commonly used
fixatives for yeasts are potassium permanganate and
osmium tetroxide, and the latter is popular for
excellent results. In particular, osmium tetroxide
provides better contrast for intracellular organelles
of fungal elements interacting with the animal or
human tissues [14]. Intracellular organelles particularly nucleoli are not clearly visible when Candida is
fixed in potassium permanganate solution [15].
Tissues and fungi for SEM are generally fixed with
osmium tetroxide prior to gold coating for final
observations.
Various staining chemicals such as uranyl acetate,
lead citrate, and ruthenium red are useful in differentiation of fungal elements within the tissues by
TEM. Ruthenium red specifically stains yeast cell
wall polysaccharides, and it is useful in detailed
visualization of the outer fibrillar layer of candidal
cell wall [16]. Interestingly, this stain has produced
contrasting visuals of extracellular polysaccharides
associated with the yeast cell wall [17]. Additionally,
alkaline bismuth staining that can be used to stain
polysaccharides in electron microscopic studies is
another substitute for ruthenium red [18].
Furthermore, cytochemical and immunohistochemical methods have also been utilized in the
exploration of the invasive phase of Candida species
on tissue samples. In a pioneering study using
cytochemical methods, Pugh and Cawson [19] examined PL activity of C. albicans in vitro. In order to
visualize localized activity of candidal PL, these
investigators introduced lecithin and lead nitrate into
the reaction medium. Lead precipitates due to
interactions with the fatty acids derived by the action

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of PL on lecithin were taken as the evidence of

enzyme activity. This study demonstrated PLs concentrated at the initial buds and the tips of candidal
hyphae. Later, this method was utilized to demonstrate localized activity of PLs in C. albicans
invading the developing chick embryo, and it was
found that PLs were present at the invading hyphal
tips denoting their cytolytic properties [20]. Moreover, using enzyme cytochemistry, De Nollin et al.
[12] have identified numerous intracellular organelles
such as nucleus, mitochondria, and secretory vesicles
in the vegetative candidal cells. Immunohistochemistry yields greater specificity since it identifies
antigenantibody reactions. Recently, immunohistochemical staining with silver- or gold-tagged antibodies has provided interesting data on candidal
SAPs [21] and PLs [22, 23].
Most of the ultrastructural studies of the genus
Candida are based on C. albicans primarily due to its
clinicopathological and epidemiological significance.
Therefore, this review mainly focuses on the ultrastructure of C. albicans interacting with diverse
mucosal sites.

Candida Ultrastructure In Vitro

Ultrastructure of Candida in vitro has been extensively explored using both TEM and SEM. Thus,
TEM observations have identified five different
electron-dense and electron-lucent layers of Candida
cell wall [24, 25]. Many investigators have attributed
such stratifications to the chemical composition of
Candida cell wall. In an extensive review on
literature, Kennedy [26] described that two polysaccharides, a-mannan and b-glucan, represent 7585%
of the dry weight of Candida cell wall, and the
remainder is composed of lesser amounts of chitin,
protein, and lipids. Mannoproteins are the major
antigenic components of candidal cell wall predominantly occupying the electron-dense areas accounting up to 40% of the cell wall. Innermost layer rich in
chitin provides cellular rigidity and predominantly
seen in hyphal phase [24]. Chitin contributes about
110% of the dry weight of Candida cell wall.
Glucans are the major constituent of electron-transparent layer while lipids are dispersed within the cell
wall accounting up to 4760% of dry weight [27].
When the cell walls of C. albicans blastospores and

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hyphae are considered by dry weight, it has been

revealed that blastospores are rich in chitin, lipids,
and carbohydrates with less amount of protein [26].
Interestingly, Howlett and Squier [28] noted five
distinct layers of C. albicans invading the rat tongue
mucosa, and it was suggested that the fungi regardless of whether intracellular or extracellular contain a
five-layered cell wall with an external fibrillar coat
during pathogenesis.
C. albicans cell membrane has a phospholipid bilayer comparable with the typical eukaryotic cell, and
other cellular organelles such as nucleus, mitochondria, and ribosomes share the features of the latter.
Cytoplasm contains several vacuoles full of secretory
components, numerous microtubules, and microfilaments such as actins and septins. Although Golgi
apparatus is not clearly identified in C. albicans,
Rajasingham and Cawson [29] have demonstrated
similar vesicular structures associated with the endoplasmic reticulum and called them as plasmalemmasomes. They also found vesicles on Candida cell wall
and named them as lomasomes. Both these plasmalemmasomes and lomasomes are thought to function
as secretory organelles of Candida species. Following
these observations, Rajasingham and Challacombe
[30] who explored the ultrastructure of C. albicans in
its invasive form within the human oral epithelium
and the chick chorio-allantoic membrane (CAM)
identified well-organized intracytoplasmic tubular
and intracytoplasmic vesicular arrangements. These
intracellular arrangements showing a variety of
configurations were named as intracytoplasmic membrane tubules (ICMT) and intracytoplasmic membrane structures (ICMS), respectively [30]. Due to
intimate relationship of the ICMS with the nucleus,
mitochondria, and plasmalemma of the invasive
forms of fungi in human and chick embryonic tissues,
it is reasonable to speculate that these structures may
be functional in cellular transport and secretory
mechanisms.
In an experiment to detect localized enzyme
activities of C. albicans grown in liquid culture, De
Nollin et al. [12] found that C. albicans blastospores
contain vacuoles rich in both alkaline and acid
phosphatases. Therefore, it was postulated that
numerous vacuoles in Candida cytoplasm indicate
high metabolic activity of the yeast in culture. Thus,

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these intracellular vacuoles may be responsible for

the cellular nutrition, growth, and the multiplication
processes. Similarly, application of amylase on
Candida cells by Rajasingham and Cawson [31]
detected numerous granules susceptible to amylase
within the yeast cytoplasm. Thus, it was concluded
that the granularity of candidal cytoplasm depicted
the abundance of glycogen that functions as nutrition
storage of mature Candida cells.
In a pioneering study using SEM, Barnes et al.
[32] explained surface features of different morphologies of Candida in vitro. They observed smooth
budding yeast cells with bud scars on the cell surface.
Initial hyphae (germ tubes) elongated to form long
filaments and mycelia while spherical chlamydospores attached to the terminal ends of some mycelia. In
addition, Barnes et al. [32] noticed some yeasts with
irregular convoluted surface structures. One of the
possible phenomena behind this could be phenotypic
switching of Candida where the organism undergoes
changes in colony morphology at different environmental conditions and growth stages [33]. In a similar
experiment, Joshi et al. [34] investigated morphological features of ten C. albicans isolates grown in
different culture media using SEM, and they found
different morphologic characteristics of Candida
including budding, filamentation, and chlamydospore
formation. Shannon [35] demonstrated using TEM
and SEM that chlamydospores of C. albicans are
large, spherical structures with bi-layered cell wall
made up of outer electron-transparent primary layer
and an inner electron-dense secondary layer. Characteristically, cytoplasms of these chlamydospores
were occupied by several vacuoles. Other cytoplasmic organelles of the chlamydospores were similar to
that of blastospores.

Ultrastructure of CandidaHost Interactions

Features of Candidahost interactions have been
explored using numerous specimens from humans
[3639], animals [28, 40], and tissue cultures [20, 41
43]. For the sake of simplicity, a number of
pathological features described in those experiments
are categorized dichotomously into virulence factors
of Candida and host responses (Table 1).

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Table 1 Important Candidahost interactions in superficial candidiasis

Virulence factors of Candida

Host responses

Adhesion and attachment

Phagocytosis/cellular internalization

Cell wall (fibrillar attachment)

Epithelial cell rupture/deformity

Cavitations/depressions
Persorption

Epithelial hyperplasia, atrophy, and dystrophy

Parakeratosis, acanthosis, and spongiosis

Morphogenesis

Lysis of tonofilaments

Blastospores, buds, true hyphae, pseudohyphae, and chlamydospores

Clear zones of cytoplasm

Thigmotropism

Loosening of gap junctions

Appressoria

Detachment of desmosomes

Irregular cells

Accumulation of PMNL and mast cells

Extracellular enzymes

Interactions with other commensals

SAPs
PL
Phynotypic switching
Rough and smooth colonies/cells

Virulence Factors of Candida

Adhesion
Adhesion to epithelial cells is an important prerequisite for the colonization of Candida especially in
environments like oral cavity where there is plenty of
factors for dislodgement, such as saliva-flushing
action, masticatory movements, and continuous epithelial renewal. King et al. [44] who examined the
adherence capabilities of different Candida species
with human oral and vaginal epithelial cells in vitro
pointed out that adherence is the initial step of
pathogenesis of superficial candidiasis. This study has
shown that Candida isolates having higher adherence
capacity are more pathogenic. It has been suggested
that a number of candidal factors as well as host
factors determine candidal adherence with the epithelial tissues. Special constituents of Candida cell
wall that facilitate adhesion are known as adhesins
[44]. These mucopolysaccharide molecules provide
mechanisms for the attachment of Candida cells with
the epithelium. Expression of such surface molecules
may be essential for the hostfungal interactions
particularly in generating the host immune responses
[45].
Many investigators using TEM studies have identified a thick floccular fibrous layer over the cell wall

of Candida attached to epithelial surfaces. In a

pioneering study, Montes and Wilborn [36] noted a
thick floccular material coating the cell wall of
Candida invading human oral epithelium. However,
the fungi internalized by the epithelium seemed to
loose this floccular material. Furthermore, it was
striking to note that the host cytoplasm at the vicinity
of intracellular fungi was clear and devoid of
tonofilaments. Therefore, it was speculated that the
floccular appearance was derived by the fungal
enzymes that could have degraded intracellular
filaments of epithelial cells in addition to providing
adhesive property to the yeast cell. Similarly, floccular appearance associated with invading fungal elements has been described in a series of experiments
using biopsies of human candidal lesions [14, 37, 38,
46, 47]. Most significantly, these floccular or fluffy
extracellular substances have often been noticed
particularly at the invading hyphal tips of Candida.
In an attempt to investigate the gastrointestinal
colonization and the systemic dissemination of
C. albicans and C. tropicalis in immunocompromised
mice, De Repentigny et al. [48] found that either
species colonize gastrointestinal epithelium by lysing
adjacent microvilli leading to progressive dissemination. Moreover, it was found that the blastospores
lying within villi were surrounded by a conspicuous
clear zone denoting cytolytic activity. Moreover, in a

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study to examine candidal invasion of female genitourinary tract, Garcia-Tamayo et al. [49] examined
exudates collected from the vagina and the uterine
cervix of ten patients suffering from genital candidiasis using ruthenium red stain. Their observations
revealed that mucopolysaccharides were present in
the glycoprotein coat of fungi lying both within and
outside of the epithelial cells. Their SEM observations also demonstrated bud scars and glycoproteinaseous substances with a floccular appearance on the
surface of the invading fungi.
Korting et al. [50] who pioneered ultrastructural
studies of candidal invasion of reconstituted epithelia
found high affinity of Candida blastospores toward
the desmosomes of the reconstituted human epidermis. These investigators surmised that Candida cells
adhere to surface epithelial cells through the surface
fibrillar layer. Recently, using ruthenium red stain,
Vitkov et al. [51] demonstrated a fibrillar extracellular coat (glycocalyx) over the candidal cell wall
attached to the human oral epithelium affected by
denture stomatitis. It was also found that the invading
blastospores are internalized by the oral epithelial
cells, and remarkably, such internalized fungal elements were lacking a fibrillar coat comparable with
the previous observations of Rajasingham and Cawson [47]. However, Vitkov et al. [51] suggested that
the extracellular fibrillar coat acted as an adhesive
apparatus (fimbria-mediated epithelial attachment)
exhibited by C. albicans during pathogenesis. In
addition, post-mortem biopsies of various parenchymal organs, including kidney, liver, spleen, heart,
esophagus, and rectum of disseminated candidiasis
patients, have demonstrated a thick fibrillar outer coat
in blastospores multiplying within the tissues [52].
These investigators have argued that the fibrillar coat
is derived from the epithelial fibrin. As such, the
exact origin and the function of the candidal fibrillar
coat should be further investigated to describe its role
in the pathogenic process.
Numerous secretions present at the epithelial
surface affect the homeostasis of the normal microbial flora. In an early attempt to explore the behavior
of Candida with gastrointestinal epithelium, Pope
and Cole [53] observed differential candidal attachments with the mouse gut epithelium after intragastric inoculation of two C. albicans isolates to infant
mice. There were Candida blastospores attached to
the keratinized stomach epithelial surface mixed with

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rod-shaped lactobacilli. In the small intestine, yeasts

were seen embedded in mucus layer, and it was
suggested that candidal attachment is facilitated by
the mucus in gastrointestinal system. Later, in a series
of experiments, Cole et al. [5456] described a
murine model using infant mice (5- to 6-day old) for
the investigation of hostparasite interactions during
gastrointestinal candidiasis. Their studies using SEM
and TEM of the murine gut epithelium affected by
C. albicans revealed that fungal adhesion and invasion are mediated by the lysis of mucous secretions.
Furthermore, SEM showed that after 1 h of oralintragastric inoculation, the yeasts were partially
embedded in microvilli layer of the intestinal epithelium while TEM studies indicated a clear zone of
cytoplasm surrounding the yeasts that had entered the
gut epithelium. These observations pointed out that
Candida possesses mucinolytic activity possibly
conferred by the extracellular enzymes.
Many investigators were of the view that candidal
adhesion altered epithelial surface so as to enhance
the attachment by preventing the dislodgement by
physical forces. For instance, in an experiment to
observe the modes of Candida adherence to rabbit
esophageal epithelium, Hoshika and Mine [57]
described four different phenomena including attachment, subepithelial insertion, cavitation, and hyphal
invasion. These investigators argued that the above
features at the fungalepithelial interface are essentially brought about by the physical contact between
yeast and the epithelial cells. Further exploration of
these findings using healthy and immunosuppressed
rabbits fed on sucrose-rich diet revealed that adherence of C. albicans to esophageal mucosa occurs
irrespective of the host immune status [58]. These
investigators suggested that subepithelial cell insertion is a primary mode of yeast adherence to
esophageal epithelium.
Ray and Payne [59] who investigated ultrastructural features of cutaneous candidiasis produced in
new-born mice with several Candida species, including C. albicans, C. stellatoidea, C. tropicalis,
C. parapsilosis, C. guilliermondii, and C. krusei,
found that C. albicans was the most virulent species
having high hyphal-forming capacity and increased
adherence with the skin corneocytes. They observed a
mucinous material facilitating fungal adherence with
the epithelium over the yeast and hyphal forms and
called them as cohesins. Most interestingly, there

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were cavitations on the corneocytes of mouse skin

complementary to fungal elements that were in
contact after 8 h of incubation. These cavitations
resulted by a large number of blastospores had given
surface epithelium a characteristic egg carton
appearance. It was suggested that those cavitations
were resulted by the proteolytic degradation of
epithelial keratins by candidal proteinases released
at the invasive interface. Their TEM observations on
the cell wall of Candida blastospores in the mouse
epithelium were comparable with previous findings
of an electron-dense fibrillar structure on candidal
cell wall interacting with the oral mucosa of
SpragueDawley rats and New Zealand white rabbits
[28]. Therefore, surface cavitations and depressions
caused by Candida possibly through the extracellular
enzyme activity seem to facilitate yeast survival and
attachment on different epithelial surfaces.
The foregoing studies show that the adhesion of
Candida with the epithelial tissue is a complex
phenomenon. Finally, it is the composite effect of the
surface fibrillar structure of yeast cell wall and the
host epithelial cell structure that facilitate candidal
adhesion. Moreover, candidal extracellular enzymes
and host-derived molecules such as mucous secretions along with the resident microbial flora play an
important role in candidal adherence to the host
epithelium. These complex mechanisms are warranted for further investigations up to the molecular
and receptor levels, since the adhesion is the crucial
stage of the serious infectious process of Candida.

Hyphal Invasion
Histopathology of mucosal candidiasis is characterized by the invasion of hyphae (true and pseudo) into
the superficial epithelium [60]. It has been demonstrated that hyphal mutants of C. albicans that do not
produce hyphae are unable to produce invasive
candidiasis in mice [61] as well as in tissue cultures
[62]. On the other hand, non-albicans Candida
species without hyphae were non-invasive in experimental human oral candidiasis produced on reconstituted human oral epithelium [63]. These
observations indicate that the conversion of blastospore into filamentous hyphae plays an important role
in candidal virulence. Ultrastructural investigations
of superficial candidiasis have pointed out that

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filamentous hyphae act as invasive appendages

during pathogenesis.
In a very early study, Montes and Wilborn [36]
examined the ultrastructure of the epithelial scales
harvested from oral candidiasis patients, and they
found fungal elements in intra and extracellular sites.
These observations were confirmed by subsequent
studies, and it has been concluded that Candida
invades intra or intercellularly within the epithelium
[39, 46, 64]. In a pioneering study, Howlett [65]
examined invasive potentials of C. albicans,
C. tropicalis, C. krusei, C. parapsilosis, and
C. guilliermondii in oral mucosal explants cultures
of rats and rabbits. This study demonstrated differential invasion of Candida species into the epithelia
lining tongue, palate, and the cheek. SEM and TEM
observations revealed that highly invasive C. albicans in both blastospore and hyphal forms penetrated
rat tongue and the rabbit buccal mucosa in vitro.
Furthermore, SEM features of chronic mucocutaneous candidiasis lesions have illustrated that predominant morphological structures during infection are
pseudohyphae [66].
Alternatively, Stoetzner and Kemmer [67] who
examined autopsy samples of humans died of candidiasis using ultrastructural methods found that Candida penetrates the esophageal epithelium in both
blastospores and hyphal forms. These fungal elements showed several intracellular organelles including vacuoles, mitochondria, and ribosomes showing
increased intracellular metabolic activity. Similarly,
ultrastructural features of skin biopsies obtained from
humans with candidiasis of groin, axilla, and submammary areas have confirmed that candidal invasion into the cutaneous tissues is mainly assisted by
the hyphae [68]. Moreover, Howlett and Squier [28]
noted that the site of hyphal invasion was distant
from the site of attachment of the parent blastospores
indicating that the candidal colonization and the
invasion are independent processes.
In the systemic pathogenesis of candidiasis, fungi
usually enter into the systemic circulation through the
endothelium. In vitro culture of porcine vascular
endothelial explants was used by Klotz et al. [69] in
order to investigate interactions between Candida
species and the endothelium. Those investigators coincubated different Candida species with the porcine
vascular strips and found that C. albicans and C.
tropicalis adhere to endothelium far better than other

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Candida species. Their SEM and TEM observations

demonstrated that C. albicans blastospores lying on
the endothelium could breach cellular integrity and
burrow deeply into the vascular tissue. There were
occasional germ tubes penetrating through the surface
endothelium of the explants [69]. Taken together,
these studies elaborate that hyphae that apply physical and biochemical pressure on host epithelia are
vital for the invasive pathogenesis of candidiasis.
Thigmotropism
Thigmotropism is a phenomenon where orientation of
growing organisms is guided by the physical contact
especially by a solid or rigid surface. Wilborn and
Montes [66] suggested that candidal hyphae show
contact sensing or thigmotropism after they observed
hyphae penetrating through the holes on oral
epithelial cells obtained from chronic mucocutaneous
candidiasis patients. Afterward, Sherwood et al. [70]
have demonstrated using nuclepore membranes that
candidal hyphae entered through surface micropores
following thigmotropic behavior. Later, this phenomenon was elegantly elucidated by demonstrating
contact guidance of candidal hyphae on various
substrates such as polycarbonate membranes, glass,
and polystyrene having pores, grooves, and ridges on
the surface, respectively [71]. Latter studies demonstrated that candidal hyphae not only detect micropores but also follow the grooves on glass slides and
ridges on polystyrene. It was confirmed that candidal
hyphae respond thigmotropically and morphologically to surface cues on which they are grown. These
observations have pointed out that thigmotropic
behavior of fungal hyphae may trigger onset of
candidiasis on breached epithelia by facilitating
adhesion and invasion through the microscopic
wound sites and surface irregularities of the epithelia.
Ultrastructural observations of epithelial biopsies
obtained from pseudomembranous oral candidiasis of
HIV-affected patients have revealed classic histological features of hyphal penetration of the superficial
epithelium. Reichart et al. [64] found that some
fungal hyphae penetrate through intercellular gaps of
the oral epithelium affected by candidiasis resembling thigmotropism. Similarly, it has been demonstrated that candidal hyphae find easy access into the
epithelium thigmotropically through intercellular
gaps in tissue culture models like reconstituted

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human oral epithelium [42] and rabbit tongue mucosal explants [43].
Appressoria
Appressoria are bulging structures that are seen at the
tips of the hyphae of some plant pathogenic fungi, and
they generate physical forces to facilitate tissue
invasion by the fungi into various substrates [72].
Ultrastructural investigations of superficial candidiasis
too have revealed bulging structures at the hyphal tips
resembling appressoria [47, 73]. These investigators
have suggested that bulging appressoria of Candida act
as pre-penetrative structures during pathogenesis. In a
comprehensive study of ultrastructural features of the
invasive phase of Candida in vaginal candidiasis,
Rajasingham et al. [73] examined sixty-three vaginal
scrapings of acute vaginal candidiasis patients and
noticed appressorial bulge at the tips of the invading
candidal hyphae. Furthermore, ultrastructure of those
appressoria indicated numerous cell organelles including mitochondria, endoplasmic reticulum, ribosomes,
and vesicles denoting an increased metabolic activity.
Hence, it was postulated that candidal appressoria
could generate increased turgor pressure at the hyphal
tips to facilitate penetration of the host epithelium
resembling some plant pathogenic fungi. Moreover,
Rajasingham et al. [73] surmised that physical forces of
the candidal appressoria act in tandem with the fungal
enzymes released at the tips of hyphae in invasive
pathogenesis. In a similar experiment, Rajasingham
[14] has demonstrated bulging appressoria at the
penetrating hyphae and associated extracellular material that superimposed the enzymatic activity in both
oral and vaginal candidiasis. In addition, some investigators have suggested that appressoria provide
anchorage effect besides acting as penetrative apparatus for invading fungi within the oral environment [64].
Extracellular Enzymes
Candida species produce several extracellular
enzymes, and the most important enzymes that are
responsible for virulence are secretory aspartyl proteinases (SAP), lipases, and phospholipases (PL).
Apart from facilitating the nutrient supply, these
hydrolases are involved in candidal: (1) invasion by
digesting host cell membranes, (2) adhesion by
degrading host cell surface molecules, and (3)

Mycopathologia (2011) 171:235250

resistance to host immunity by attacking the immune

system [74]. Many ultrastructural studies have demonstrated localized tissue destruction in association
with the invading fungal elements. For example,
Howlett and Squier [28] found the destruction of the
oral mucosa of SpragueDawley rats and New
Zealand white rabbits by invading C. albicans and
surmised that localized tissue destruction is caused by
extracellular enzymes of Candida. Using SEM, Farrel
et al. [75] demonstrated that C. albicans invasion of
cultured human vaginal epithelial cells began after
2 h of inoculation with the appearance of characteristic flocculant material in relation to invading
hyphae, which could be attributed to extracellular
enzyme activity. However, precise distribution of
various enzymes in relation to fungal elements
interacting with the host tissues is not fully resolved.
Yet, enzyme histochemistry [21, 76] and cytochemistry [42] have shown satisfactory results in identification of some enzyme activities of Candida.
Secretory Aspartyl Proteinases (SAP)
Since the first evidence of proteolytic activity of
Candida [77], many investigators have attributed
candidal virulence to its proteinases. At present, ten
different secretory aspartyl proteinases (SAP) of C.
albicans have been described, and similar proteinases
are produced by other non-albicans Candida species
[78]. Production of SAP by C. albicans and their
involvement in invasion have been assessed using
numerous candidiasis models. In a very early study
using immunoscanning electron microscopy, Borg
and Ruchel [79] demonstrated candidal proteinases
on the surface of invading fungal elements on the
human buccal epithelial explants. They noted that C.
albicans and C. tropicalis are highly proteolytic than
other species. It was suggested that SAPs were
secreted out, as they were dispersed on the surface of
fungi and the tissue. Similarly, Kobayashi et al. [80]
using chick chorio-allantoic membrane (CAM) model
pointed out that SAPs are involved in invasive
pathogenesis of candidiasis. Their TEM observations
have demonstrated that SAP-producing C. albicans
penetrates the mesoderm layer of the CAM through
the ectoderm unlike SAP deficient isolates. In addition, application of pure candidal proteinase extract
into CAM showed loosening of the intercellular gaps
and damaged the triple layers: ectoderm, mesoderm,

243

and endoderm. Most interestingly, Kobayashi et al.

[80] pointed out that the treatment of CAM with
purified proteinases allowed invasion of proteinase
deficient C. albicans elucidating the involvement of
candidal proteinases in the pathogenesis.
Strikingly, immunoelectron microscopic studies
have revealed differential expression of SAP proteins
during experimental and clinical oral candidiasis [21,
76]. These investigators found that SAP1-3 are highly
localized on the yeast cell wall whereas SAP46 on
the hyphae. In a reconstituted human oral epithelium
model infected with C. albicans for 48 h, Schaller
et al. [76] using post-embedding labeling with goldconjugated SAP antibodies observed regular distribution of SAP within the yeast cytoplasm and the
damaged epithelial cells. In a subsequent attempt,
Schaller et al. [21] using immunoelectron microscopy
with pre- and post-embedding with silver-enhanced
labeling demonstrated that SAP13 are produced by
Candida in HIV-associated pseudomembranous oral
candidiasis. These investigators found that SAP13
were concentrated within Candida cells invading oral
epithelium depicting their involvement in pathogenesis. Furthermore, the same group of investigators,
Schaller et al. [41, 81] using post-embedding immunoelectron microscopy showed that SAP13 were
predominantly expressed on cell walls and the
cytoplasm of C. albicans blastospores, whereas
SAP46 were localized at the tips of hyphae during
oral and cutaneous candidiasis. On the whole, these
data suggested differential pathogenic roles for
SAP13 and SAP46 during hostfungal interactions
in vivo.
Phospholipases (PL)
Phospholipases (PL) in general are important proteins
that attack phospholipids of the cell membranes.
They are found in many bacterial toxins, arthropod,
and snake venoms and well known for their cytolytic
activity. These enzymes are classified into phospholipase A, B, C, D, lysophospholipase, and lysophospholipase-transacylase according to their mode of
action and the target molecules [23]. Strikingly,
C. albicans is reported to produce most of these
enzymes during pathogenic processes. For instance,
Barratt-Bee et al. [82] examined PL activity of yeasts
including C. albicans and C. parapsilosis and
assessed their virulence attributes such as adhesion

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to buccal epithelial cells and lethality in mice after

intravenous inoculation. These experiments demonstrated positive correlations between PL activity and
fungal virulence proving it as an important factor in
candidal virulence.
In a pioneering study, Pugh and Cawson [20] using
chick chorio-allantoic membrane (CAM) model elegantly demonstrated that PL was actively involved in
the invasive phase of C. albicans. These investigators
used cytochemical methods to visualize PL activity
and found that the enzyme was localized at the tips of
invading hyphae and the initial sites of bud formation. They also noted that the advancing hyphae
penetrated superficial cells of CAM rupturing latters
cell membranes and disrupting the cytoplasm leaving
a clear zone surrounding the internalized yeasts. This
effect was thought to bring about by the lytic
enzymes released by the yeast. In a subsequent
study, Rajasingham and Cawson [47] infected CAM
with Candida and compared ultrastructure with that
of the human oral candidiasis. They noted a floccular
appearance quite prominently associated with the
invading hyphae. These investigators attributed such
floccular material to extracellular enzymes of the
fungi. Recently, these studies have been extended to
tissue culture models, and Jayatilake et al. [42]
reconfirmed cytochemically that phospholipases are
expressed at the tips of candidal hyphae and initial
buds of C. albicans during invasion of reconstituted
human oral epithelium.
Furthermore, specific detection of candidal PLs
during pathogenesis has been performed using immunohistochemistry. For example, Ghannoum [23]
demonstrated that PLB was localized at the advancing tips of candidal hyphae during invasion of murine
gastrointestinal tissues. Furthermore, PLB immunogold particles were detected dispersed within mice
tissues suggesting that the enzyme PLB was secreted
out during candidal infection despite expression on
the cell walls of the invading organisms [23]. Another
experiment that examined kidneys of mice infected
systemically with C. albicans has demonstrated
distribution of candidal PLB within kidney parenchyma [22]. Those observations were also comparable with the hypothesis that PLB is predominantly
localized at the penetrating candidal hyphae. Collectively, these studies confirm the fact that PLs of
Candida are involved in the pathogenesis of candidiasis by facilitating the tissue penetration.

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Phenotypic Switching
Triggered by numerous environmental factors, Candida species undergo random alterations in colony
morphologies. This process includes an epigenetic
change of colony morphologies and precisely known
as phenotypic switching [83]. For the first time,
Slutsky et al. [33] observed spontaneous switching of
colony phenotypes (star, ring, irregular wrinkle, hat,
stipple, and fuzzy) of C. albicans grown on amino
acid-rich agar, and it was surmised that phenotypic
switching has a direct effect on candidal virulence.
Furthermore, ultrastructural observations of white
and opaque phenotypes of C. albicans have revealed
structural and morphological changes of the yeast cell
organelles including the nucleus suggesting that
phenotypic switching could affect the behavior of
the organism [84]. However, phenotypic switching
differs from morphogenesis (yeast-hyphal transition),
as it is assigned to describe the spontaneous morphological changes of the colonies of growing
Candida on solid media [85]. Finally, it has now
been proposed that switching as a mechanism of
accelerated microevolution allowing survival of the
fittest in new microniches of the growth environment
[11].
However, the relevance of phenotypic changes of
C. albicans with the Candidahost interactions has
not been well established. In an experiment to
determine the relationship between phenotypic
switching and the candidal adhesion to porcine
buccal epithelial cells using SEM, Vargas et al. [86]
demonstrated that smooth yeast colonies are more
adherent compared with irregular wrinkle, star, and
revertant smooth phenotypes. Their investigations
also revealed that differential fungalepithelial cell
interactions were based on the phenotypes. However,
further studies are necessary to ascertain the links
between phenotypic switching and the pathogenesis
of candidiasis.

Host Responses
Combinations of innate and adaptive immune mechanisms are known to act synergistically in candidiasis
[87]. Ultrastructural investigations using Candidaaffected tissue specimens have enormously helped
identify multiple host responses at the invasive

Mycopathologia (2011) 171:235250

interface of fungi and the host. Interestingly, a series

of ultrastructural studies using mucosal biopsies of
oral candidiasis lesions have elaborated that epithelial
invasion of Candida is supported by physical and
biochemical (enzymatic) forces of the fungi [14, 37,
38, 47]. In the mean time, these studies have found
that epithelial cells and immune cells do react against
the fungal challenge. First, these studies have demonstrated deformation and rupture of host cell
membranes in the presence of candidal challenge.
Usually, epithelium responds with changes such as
parakeratosis, acanthosis, and spongiosis at the
vicinity of invading candidal hyphae [68]. Moreover,
epithelial and endothelial cells have shown to engulf
yeasts resembling phagocytic cells. In addition,
submucosal tissues and the immune system respond
differentially against invasion by Candida species.

Phagocytosis
Histopathology of mucosal candidiasis is characterized by fungal invasion of the epithelium in both
blastospore and hyphal forms. On the other hand,
there are huge aggregations of polymorphonuclear
leukocytes (PMNL) within and underneath the epithelium leading to microabscess formation in mucosal candidiasis [88]. At present, it is well accepted
that PMNL and macrophages engulf invading fungal
elements as a part of normal protective mechanism.
In an early experiment to observe interaction between
C. albicans and human PMNL, Belcher et al. [89]
have demonstrated phagocytosis of Candida yeasts
by the PMNL cells. Besides, they noticed numerous
lysosomes of PMNL participate in candidal killing.
This study has also shown that single PMNL could
engulf multiple yeasts. Most interestingly, Belcher
et al. [89] pointed out that PMNL engulf yeast cells
and form phagolysosomes within 1560 min in vitro.
These observations were later reconfirmed by Marrie
and Costerton [17] who demonstrated phagocytosis
of yeasts by PMNL in pathological samples collected
from both oral and urinary candidiasis patients. In an
attempt to visualize the process of yeast phagocytosis
by macrophages, Kaposzta et al. [90] noticed pseudopodia formation by mouse macrophages to trap
Candida blastospores regardless of latters viability.
It was also confirmed that resulting phagosomes
rapidly attracted endosomes and lysosomes to form

245

phagolysosomes akin to the observations of Belcher

et al. [89].
In addition to PMNL, epithelial and endothelial
cells have also shown to behave as phagocytes of
Candida. For instance, in a very early study, Schnell
et al. [91] who examined vaginal swabs under TEM
found Candida in blastospores and buddings lying
within epithelial cells. These investigators argued that
internalized yeasts could multiply within epithelial
cells. Thus, Schnell et al. [91] speculated that
intracellular yeasts could lead to recurrent infections,
as they act as reservoirs of the pathogen unharmed by
antifungals and the immune system. Complementary
to these observations, ultrastructural studies by
Calderone et al. [92] on the interactions of
C. albicans and human buccal and vaginal epithelial
cells found that either cell type could engulf yeasts.
Furthermore, Enache et al. [93] who examined C.
albicans adhesion to human esophageal cell cultures
using TEM observed pseudopodia formation by the
esophageal cells toward the adjacent yeast cells
exhibiting a phagocytic event. Moreover, entrapped
yeasts were seen in an infolding of keratinocyte cell
membrane as observed by SEM [93]. Similarly, in an
experiment to investigate interactions of human
buccal, vaginal, and HeLa cells with Candida yeast
have revealed that all three cell types can actively
internalize yeast cells in vitro [94]. Moreover,
features of yeast internalization have been observed
in ultrastructural studies of experimental human oral
candidiasis established on reconstituted human oral
epithelium [42]. Taken together, foregoing studies
provide ample evidence of phagocytosis of Candida
by different types of epithelial cells in addition to
PMNL. Non-phagocytic cells such as epithelial cells
may harbor the yeast unharmed by the host immune
mechanisms and the effects of the therapeutics. This
has a great clinical significance, as intracellular yeast
cells can trigger recurrence of infection and antifungal resistance formation.
In the context of disseminated candidiasis, yeasts
enter peripheral tissues via blood circulation, and the
endothelial phagocytosis could facilitate this event. In
a pioneering study, Filler et al. [95] elegantly
demonstrated that human endothelial cells engulf
C. albicans yeasts in vitro regardless of their
viability. These investigators noted that out of three
Candida species, C. albicans, C. tropicalis, and C.
glabrata, endothelial cells engulf only C. albicans

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246

cells showing that endothelial phagocytosis is a

selective mechanism. It was also found that some of
the ingested Candida yeasts follow trans-cellular
passage (transmigration) through the endothelial cells
by either way: by opened vacuoles or by germinating
within the cell causing endothelial cell injury. Latter
observations were comparable with that of Kaposzta
et al. [90] who reported that internalized Candida
cells form germ tubes to evade murine macrophages
in vitro. Furthermore, in an early study, intercellular
migration (persorption) of Candida has been
described in gastrointestinal candidiasis [96]. Such
tissue penetrations provide yet another mode of
candidal invasion. As such, persorption could help
Candida entry into the blood stream and reach vital
organs like brain, heart, and kidney leading to fatal
conditions.
In another experiment to investigate Candida
endothelial cell interactions, Zink et al. [97] incubated C. albicans yeasts with bovine aortic endothelial cell monolayers. This investigation unraveled
multiple steps in trans-endothelial migration of
C. albicans. At the initial stage, pseudopodia were
formed by the endothelial cells to capture adjacent
yeasts into phagocytic vacuoles. Thus, entrapped
yeasts evaded endothelium breaking cellular integrity
by formation of hyphae. Eventually, it was found that
germ tube forming C. albicans enhanced transendothelial migration and caused more damage to
endothelial cells with reference to a hypha deficient
mutant isolate. Therefore, the trans-migration of
Candida through the endothelial cells is likely
decided by endothelial phagocytosis and the virulence attributes of yeast such as adhesion and
filamentation.
Furthermore, clinical and histopathological evidence of hyperplastic candidiasis where there is
marked epithelial hyperplasia has suggested that
candidal invasion induces epithelial hyperplasia
[98]. For instance, Nagai et al. [99] who examined
epithelial biopsies of oral candidiasis lesions using
TEM have demonstrated intricate arrangements of
dense tonofilaments and zipper-like desmosomal
junctions in the hyperplastic or dysplastic epithelium
into which fungal elements were penetrating. Interestingly, Nagai et al. [99] found that spinous cells in
the hyperplastic epithelia in oral candidiasis contained many small mitochondria in various growth

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Mycopathologia (2011) 171:235250

stages denoting increased intracellular activity.

Therefore, it was suggested that epithelial hyperplasia
is a protective mechanism of the epithelium in
response to candidal challenge and needs further
investigations.
In a comprehensive study to examine the ultrastructure of established human oral candidiasis,
Mohamed [39] used biopsy specimens of human oral
candidiasis lesions of the tongue and the lip. In this
study, subepithelial microabscesses formed by the
accumulation of PMNL and cytoplasmic dissolutions
produced by the disappearance of tonofilaments of
the epithelial cells were also detected. Interestingly,
Mohamed [39] described some of the features of the
subepithelial connective tissue response in oral
candidiasis and suggested that oral candidiasis is
accompanied by an inflammatory response predominated by neutrophils, macrophages, and mast cells.
This inflammatory response also consisted of marked
subepithelial vascular leakages through the intercellular gaps of the endothelium. These observations
testify that inflammatory reaction is also an important
part of the host response in superficial candidiasis.
Interaction with the Commensal Flora
Commensal flora of various body niches prevent
infections by competing with the pathogens as well as
inducing the immune system. Being an obligate
human commensal, Candida species do interact with
the rest of the commensal flora, and these interactions
have been described by many investigators in pathological samples. In an early study, Wilborn and
Montes [66] who examined epithelial biopsies of oral
candidiasis using SEM indicated that the invading
fungal elements had intimate interactions with the
oral bacterial flora. Moreover, Marrie and Costerton
[17] noticed numerous bacteria attached to yeast cell
walls in oral and urinary candidiasis and pointed out
that Candida interacts with other members of the
commensal flora in health and disease. However,
exact mechanisms of the rest of the normal flora that
govern candidal growth on various epithelial niches
are still shrouded in mystery. Therefore, further
studies on Candida interaction with the normal
commensals at different sites of the human body
are essential to ascertain intermicrobial interactions
in superficial candidiasis.

Mycopathologia (2011) 171:235250

Conclusions
Extensive research over the past decades on the
ultrastructure of superficial candidiasis has elicited a
variety of fungalhost interactions at the invasive
interface. Putative virulence attributes of Candida are
broadly assigned to adhesion, morphogenesis, production of hydrolytic enzymes, and phenotypic
switching. Epithelial and immune cells of the host
respond differentially to candidal challenge in the
mucosal environment full of commensal organisms.
Ultrastructural investigations are tremendously useful
in gaining more insights into these pathological
mechanisms (Table 1).
Cell wall of Candida plays an important role in its
survival on various epithelia. Its unique structure with
polysaccharide fibrils and hydrolytic enzymes
enhances resistance against dislodging forces of
physical and chemical nature in the host niches. This
fibrillar coat arising from the cell wall is a significant
feature of yeasts in close proximity with the epithelial
cells. Epithelial cells show cavitations or depressions
in relation to yeasts contact. These phenomena are
attributed to the activity of lytic enzymes such as
SAP and PL of the invading fungi. Phagocytosis of
yeasts by epithelial as well as immune cells is
important in arresting the spread of infection. Yet,
unharmed fungal elements surviving within the
phagocytic cells may facilitate recurrence of infection, as the intracellular organisms can reactivate
when the suitable conditions supervene. Most of the
ultrastructural studies focused on fungalhost relationships in superficial candidiasis testify the importance of hyphal formation by Candida in invasive
pathogenesis. Hyphae act as invasive apparatus for
fungi by generating physical forces and lytic
enzymes. Moreover, they are capable of detecting
suitable sites for invasion such as breaches and
micropores in susceptible epithelia by a process
known as thigmotropism. Formation of appressoria at
the hyphal tips further provides penetrative appendages for the pathogen. Many ultrastructural studies
have shown epithelial tissues respond to these
pathogenic stimulants by cell rupture and alterations
of the tissue integrity.
One of the most significant findings of the
ultrastructural studies on superficial candidiasis is
the involvement of extracellular enzymes at the
Candida invasive front. In early studies using SEM

247

and TEM, this extracellular enzyme activity has been

identified as extracellular floccular material on fungal
elements that became less pronounced after getting
into host cells. This effect has now been attributed to
the extracellular enzyme activity of fungi that helps
tissue penetration [14]. Cytochemistry and immunohistochemistry have revealed that these enzymes such
as SAP and PLs are localized at the invading tips of
candidal hyphae and buds depicting their association
in invasive pathogenesis. On the other hand, these
enzymes are secreted out into the tissues that are
affected by the fungal challenge. Phenotypic switching, the alteration of the colony morphologies of
Candida under different conditions, is also considered an important virulence attribute. Although many
investigators have performed ultrastructural observations of the colony morphologies in vitro, only a few
studies have focused on phenotypic changes of
Candida on different tissue surfaces. Certain phenotypes like smooth colonies seem to be highly virulent.
Moreover, various immuno-inflammatory processes
such as phagocytosis, inflammation, cellular, and
epithelial tissue changes occur at the fungalhost
interface. In conclusion, ultrastructural studies of
superficial candidiasis have disclosed numerous host
parasite interactions at the fungalepithelial interface
where Candida transforms into an opportunistic
pathogen from an innocuous commensal organism.

Areas for Future Research

Ultrastructural studies have been instrumental in
demonstrating direct contribution of candidal virulence like adhesion, morphogenesis, and the production of extracellular enzymes in pathogenesis.
However, further investigations into the molecularand receptor-level interactions of those virulence
mechanisms with the host tissues would be supportive in finding treatment modalities such as host
immuno-inflammatory modulation. It may also unravel novel drug targets. Moreover, many ultrastructural studies of superficial candidiasis have focused
more on fungal characteristics than the host response.
It is extremely vital to undertake further studies to
scrutinize the host response in superficial candidiasis
with reference to local epithelial changes and the
immune responses. In particular, exploration of yeast
internalization by non-phagocytic epithelial cells and

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248

interactions with the commensal flora may help find

new insights into the pathogenesis and recurrence of
the infection.
Furthermore, disparity in pathogenic behavior
exists among Candida species while ultrastructural
studies have focused mainly on the commonest
pathogen, C. albicans. This shows the necessity of
investigations regarding fungalhost interactions
caused by Candida species other than C. albicans,
as the latter species are emerging as pathogens
particularly among compromised populations. On the
other hand, some of the previous ultrastructural
studies are based on the established candidiasis
lesions obtained from pathological samples. Using
different candidiasis models based on either animals
or tissue cultures will facilitate detection of pathogenic process beginning from the healthy tissue in a
phase-specific manner. Thus, prudent choice of
models may be important to visualize chronological
ultrastructural pathology of superficial candidiasis by
future investigators.
Acknowledgments Author would like to thank Professor
L.P. Samaranayake, Faculty of Dentistry, The University of
Hong Kong for his constructive advice in preparation of the
manuscript.

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