Keywords
Candida albicans - oral candidiasis - microflora
Introduction
Oral candidiasis (also called candidosis) is an opportunistic infection affecting
the oral mucosa. These lesions are very common and caused by yeast Candida albicans. C. albicans are normal commensal of oral microflora and more than 30% of individuals carry these
organisms. The rate of carriage increases with advancing age of the patient and C. albicans has been recovered from oral cavity of most of the adults with bad oral hygeine.[1]
[2]
The organism under favorable condition has the ability to transform into pathogenic
hyphal form. Conditions that favors this transformation include use of broad spectrum
antibiotic therapy, xerostomia, immune dysfunction, overuse of antibiotics, rise of
acquired immunodeficiency syndrome, increase in organ transplantations and the use
of invasive devices, diabetes, and presence of removable prosthesis.[3]
Other species such as C. glabrata, C. tropicalis, C. guilliermondii, and C. krusei are infrequently isolated from various clinical specimens.
Common Species of Candida
Common Species of Candida
Candida albicans
It is the predominant cause of invasive fungal infections. People at risk include
those suffering from human immunodeficiency virus (HIV), cancer, and intensive care
unit patients who are undergoing major surgery and organ transplants.[3]
Candida glabrata
It has become important because of its increasing incidence worldwide and decreased
susceptibility to antifungals. Its emergence is largely due to an increased population
of immune compromised patient and wide spread use of antifungal drugs. In many hospitals,
C. glabrata is the second most common cause of candidemia.[4]
Candida tropicalis
It is the third or fourth most commonly recovered Candida species from blood cultures.
C. tropicalis has progressively been observed to be the most common cause of invasive candidiasis
in neutropenic patients such as those with acute leukemia or those who have undergone
bone marrow transplantation.[5]
Candida parapsilosis
It is one of the principal causes of invasive candidiasis. In most parts of the world,
it is the third most common cause of candidemia especially in patients with intravenous
catheters, prosthetic devices, and intravenous drug use. It is also one of the most
common causes of candidemia in neonatal intensive care unit.[6]
Candida krusei
It is the fifth most common bloodstream isolate, although less common (1–2%). C. krusei is of clinical significance because of its intrinsic resistance to fluconazole and
reduced susceptibility to most other antifungal drugs. It is frequently recovered
from patients with hematological malignancies complicated by neutropenia and tends
to be associated with higher mortality rates (49 vs. 28% with C. albicans) and lower response rates (51 vs. 69% with C. albicans).[7]
Candida guilliermondii
It has been isolated from environmental surfaces and from the skin and nails of health
care workers. It has been shown to cause hematogenously disseminated candidiasis.[8]
Proposed Revised Classification of Oral Candidosis[9]
Proposed Revised Classification of Oral Candidosis[9]
Primary Oral Candidosis (Group 1)
Acute symptoms are classified into pseudomembranous and erythematous and chronic symptoms
are classified into erythematous, pseudomembranous, hyperplastic, nodular, and plaque-like
Candida-associated lesions, which include angular cheilitis, denture stomatitis, and
median rhomboid glossitis.
Keratinized primary lesions superinfected by candida are leukoplakia lichen planus
and lupus erythematosus.
Secondary Oral Candidoses (Group 2)
Oral manifestations of systemic mucocutaneous candidiasis are due to diseases like
thyroid aplasia and candidiasis endocrinopathy.
Pathogencity of Candida Species[10]
The factors involved in pathogenicity of C. albicans have been reviewed. The pathogenesis of different biotypes and strains of C. albicans varies.
Enzymes of Candida
It has been suggested that C. albicans produces endotox-in, but the levels of endotoxin produced in vivo may not be sufficient
to produce toxic effects. Alternatively, the organisms may produce enzymes that facilitate
penetration of the mucous membrane. Candida certainly have the ability to produce
phospholipases and these are concentrated at the tip of the fungal hyphae and localized
in the vicinity of the host cellular compartments where active invasion occurs. These
enzyme activities were found in most C. albicans strains but not in organisms known to be less virulent than C. albicans such a C. glabrata, C. tropicalis, and C. parapsilosis.
Extracellular proteinases have also been implicated in the pathogenicity of C. albicans. Proteinase-deficient strains are noninvasive, and pattern of adherence also reflects
the expression of secretory proteinase. Salivary proteins including immunoglobulin
A (IgA) can be almost completely degraded by acidic proteinases of Candida especially
under low pH conditions. Recently, it has been shown that parotid saliva is more resistant
to the proteolytic action of Candida proteinase when compared with mixed saliva. Acid
proteinase (aspartyl proteinase) production is increased by C. albicans isolated from later stage of HIV infection and may contribute to candidosis.[11]
Temperature Variations
The virulence of C. albicans can also be influenced by the temperature at which it is grown. Virulence is associated
with increased germ tube production by yeast grown at lower temperature. These in
turn display enhanced adherence characteristics compared with parent yeast mainly
in the blastospore phase. Yeasts grown at room temperature are more resistant to killing
by polymorphonuclear leukocytes.
Adhesion of Candida[12]
[13]
A relationship has been suggested between the adherence of C. albicans to surfaces and its ability to colonize and cause disease. An important aspect of
the pathogenicity of C. albicans may be its nonspecific affinity and binding to acrylic resin and other plastics.
The mechanism of attachment is believed to involve the interaction of cell wall components
of C. albicans with the target surface.
Factors Affecting Adhesion of Yeasts[14]
[15]
Factors related to yeast cells are medium in which it is grown, its phenotype, and
capacity to form germ tube or pseudohyphae extracellular polymeric material such as
flocculator fibrillar surface layers mannan, chitin, hydrophobicity, and cellular
lipids. Factors related to host cells are cell source, mucosal cell size and viability,
fibronectin, fibrin, sex hormones, and yeast carriers versus patients with overt candidosis.
Environmental factors affecting adhesion are cations, pH, sugars, saliva, humoral
antibody and serum, antibacterial drugs, and lectins.
Switching Phenomenon[13]
C. albicans frequently exhibits variant colonial forms when grown in vitro. A smooth colony forming
yeast when inoculated onto an agar surface may produce a proportion of colonies with
rough surfaces. It is known that switching can be triggered by low doses of ultraviolet
radiation, and once triggered into the high-frequency switching mode, C. albicans exhibit high rates of alterations in colony morphology. Thus, C. albicans has the capacity to switch frequently and reversibly between several variants, heritable,
and phenotypes. Switching is associated with change in micromorphology, and physiological
properties as well as several putative virulence traits. One switching system, “white-opaque”
transition has been examined for the capabilities of two phenotypes to adhere to oral
epithelial cells.
There are three general factors which helps the C. albicans infection to develop in the patient’s body. They are immune status of the patient,
oral mucosal environment, and strain of C. albicans.
The main factors which increase the susceptibility of oral candidiasis are[14] immunosupression, endocrinopathies, nutritional deficiency, malignancies, dental
prosthesis, epithelial alteration, high carbohydrate diet, infancy and old age, poor
oral hygiene, and heavy smoking.
Xerostomia saliva contains IgA which inhibits binding of C. albicans to mucosal surfaces. It also provides a flushing action which removes C. albicans from the oral cavity. In case of xerostomia, both these actions are absent because
of lack of saliva production, so chances of candidiasis is more in oral cavity. Xerostomia
is also seen in case of anticancer treatment and irradiation which increases the proliferation
of candidal cells and resistance of candidal cells to antifungal drugs. Xerostomia
is also seen in case of Sjogren’s syndrome because of lymphocytic infiltration and
destruction of salivary glands.[16]
Diabetes Mellitus
Growth of C. albicans thrives on increased levels of glucose in saliva which increases the ability of C. albicans to adhere to oral mucous membranes.
Medicines
Prolonged use of antibiotics depletes normal oral flora and enables proliferation
of C. albicans in the oral cavity. In asthmatic patients due to use of steroid inhalers, steroid
aerosols interfere with the normal balance of microflora and favor the proliferation
of C. albicans, whereas systemic steroids cause suppression of the Candida.
Pseudomembranous Candidiasis
Pseudomembranous candidiasis or oral thrush is the most commonly diagnosed and most
easily recognizable form of oral candidiasis. In this type of infection, the mucosa
is covered in a white or yellow pseudomembrane consisting of fibrin, desquamated epithelial
cells, inflammatory cells, and sometimes bacteria or food debris.[14] The plaque is also heavily infiltrated by fungal hyphae. With some pressure, the
membrane can be removed and underneath the mucosa is erythematous which is inflamed.
If removal of the membrane reveals bleeding mucosa, the patient is most likely suffering
from additional conditions such as erosive lichen planus or pemphigus, which are often
associated with oral candidosis in affected areas.
The infection can often be asymptomatic and other times the patient will describe
discomfort such as burning sensation, tenderness, or changes in taste when large parts
of the mucosa are involved. Most commonly affected are the buccal mucosa, tongue,
soft palate, and oropharynx.
Erythematous Candidiasis[15]
Erythematous candidiasis is probably the most common form of oral candidiasis. Due
to its less pathognomonic appearance, however, it is not as easily diagnosed as its
pseudomembranous counterpart. Erythematous candidiasis appears as red, more or less
circumscribed lesion in the hard palate or dorsum linguae. An ill-fitted denture is
also a contributing factor, as repeated trauma against the mucosa can cause an increase
in penetration of Candida antigens and toxins. The lesion is restricted to the mucosa
covered by the denture and is typically asymptomatic.
When erythematous candidiasis affects the tongue, a smooth red patch appears where
the filiform papillae atrophy. If this patch is round or oval, and is located in the
middle of the tongue, it is called median rhomboid glossitis or central papillary
atrophy. This type of lesion may be caused by bacteria as well as Candida and other
fungi, so the etiology is not completely clear. Predisposing factors for this particular
type of lesion is smoking and the use of steroid inhalers.
Linear Gingival Erythema
Linear gingival erythema is a specific type of oral fungal infection that most frequently
affects patients with HIV. The lesion is red band stretching along the gingival margin,
and can be mistaken for gingivitis. Though the diagnostic criteria are not entirely
clear, linear gingival erythema is defined as a nonplaque-induced gingivitis presenting
a distinct erythematous band of at least 2 mm along the margin of gingivae, with either
diffuse or punctuate erythema of the attached gingivae. Improved oral hygiene, even
with regular professional cleaning, is not an efficient treatment. The fungus Saccharomyces cerevisiae, Candida dubliniensis, and opportunistic bacteria are thought to be the pathogens associated with this
type of lesion.
Hyperplastic Candidiasis
Hyperplastic candidiasis is the least common form of oral candidiasis, but its malignant
potential makes it an important one. It is also called candidal leukoplakia, and like
ordinary leukoplakia, it appears as white lesion that cannot be rubbed off. Its appearance
varies greatly, from small translucent, slightly raised lesions, to large, plaque-like
areas that feel hard and rough on palpation. It is most commonly found on the buccal
mucosa and is associated with smoking. Though it cannot be rubbed off, it can be separated
from leukoplakia by microbiological tests, attempting treatment with antifungal medicine,
or by taking a biopsy for histological examination. The fungi’s hyphae often invade
the oral epithelium which is hyperplastic. As previously mentioned, the lesion of
hyper-plastic candidiasis can sometimes turn malignant, but there is controversy regarding
the importance of Candida species as a contributing risk factor.
Angular Cheilitis
Angular cheilitis is a multifactorial condition that can be caused by bacteria, especially
Staphylococcus aureus, fungi, or combination of both. It is also affected by the loss of vertical dimension,
vitamin B12 deficiency, and iron deficiency anemia. The connection between folic acid
deficiency and angular cheilitis was made in 1971 by J.A Rose, who found a significantly
higher occurrence of folic acid deficiency in patients with angular cheilitis. Folic
acid therapy was also found to heal the lesions, though this occurred in only two
of the patients, and thus was not conclusive.
Angular cheilitis affects the corners of the mouth and the surrounding skin and mucosa.
Folds in the skin create a constantly moist environment, with perfect growth conditions
for both bacteria and fungi. The result is a red, sensitive lesion, with fragile skin
that can rupture when stretched, such as when opening the mouth wide during dental
treatment. Treatment of the fungal infection will often cure the lesion, but if the
vertical dimension is not improved (denture relining), or the nutrient deficiencies
are not treated, the lesions will most likely recur.
Laboratory Diagnosis of Oral Candidiasis
Laboratory Diagnosis of Oral Candidiasis
Specimen Collection[17]
The specimen should be collected from any part of active lesion. Sufficient specimen
under aseptic precautions should be collected. Sterile collection devices and containers
should be used. The specimen should be appropriately labeled. All clinical specimens
should be handled with care using standard precautions.
The specimen should be sent immediately or stored in a refrigerator at 4°C. Due to
variety of clinical forms of oral candidiasis, several different types of specimen
may be submitted to the laboratory.
Smear
Smears are collected from infected oral mucosa, rhagades, and fitting side of denture,
preferably with wooden spatulas. Smears were fixed immediately and stained by Gram
stain method and Gridley’s periodic acid-Schiff (PAS) technique. Yeast cells appear
dark blue in Gram stain and red in PAS preparation.
Swabs
Swabs are collected from any part of the lesion inoculated on Sabouraud’s dextrose
agar incubated at 25°C and on blood agar at 37°C. Incubation at 25°C is done to ensure
recovery of species growing badly at 35°C. Since mixed yeast infections are seen in
oral cavity more frequently, particularly in immune compromised and debilitated patients,
Pagano– Levin agar or Littmann substrate are useful supplements because they enable
distinction of yeasts on the basis of difference in colony color.
Biopsy
Biopsy specimen should be taken for histopathological examination when chronic hyperplastic
candidiasis is suspected.
Imprint Culture Technique[18]
This technique uses a sterile plastic foam pad of known size (2.5 × 2.5 cm), dipped
in Sabouraud’s broth, and placed on the restricted area under study for 30 to 60 seconds.
Thereafter, the pad is placed directly on Pagano–Levin or Sabouraud’s agar, and left
in situ for the first 8 hours of at 37°C. Then, the candidal density of each site
is determined by a Gallenkamp colony counter and expressed as colony-forming units
(CFUs) per mm[2]. Thus, it yields yeasts per unit mucosal surface. It is useful for quantitative
assessment of yeast growth in different areas of the oral mucosa and is thus useful
in localizing the site of infection and estimating the candidal load on a specific
area.
Impression Culture Technique[15]
This method is totally a research tool and is useful in quantifying the relative distribution
of yeast on oral surfaces such as teeth and palate. Maxillary and mandibular alginate
impressions are taken and transported to the laboratory and casting is done in 6%
fortified agar with incorporated Sabouraud’s dextrose broth. The agar models are then
incubated in a wide-necked sterile screw topped jar for 48 to 72 hours at 37°C and
the CFU of yeasts estimated.
Salivary Culture Technique
This simple technique involves requesting the patient to expectorate 2 mL of mixed
unstimulated saliva into a sterile universal container, which is then vibrated for
30 seconds on a bench vibrator for optimal disaggregation. The number of Candida expressed
as CFU/mL of saliva is estimated by counting the resultant growth on Sabouraud’s agar
using either the spiral plating or Miles and Misra surface viable counting technique.
The carriers and patient with oral candidiasis can be distinguished reliably on the
basis of quantitative culture. Patients who display clinical signs of oral candidiasis
usually have more than 400 CFU/mL.[19]
Oral Rinse Technique
It was described by Samaranayake.[20] The concentrated oral rinse culture technique has advantages over imprint technique.
It is simple to perform and it does not involve the clinicians in judgment of sampling
size.
Paper Points
An absorbable sterile point is inserted to the depth of the pocket and kept there
for 10 seconds and then the points are transferred to the transport medium, which
also facilitates survival of facultative and anaerobic bacteria.
Commercial Identification Kits
Rapid commercial system such as Microstix-Candida and Oricut-N were used for diagnosis
of oral candidosis in the clinical setting particularly when microbiology laboratories
are not within easy access.[21]
Histological Identification
Demonstration of fungi in biopsy specimens may require several serial sections to
be cut. Fungi can be easily demonstrated and studied in tissue sections with special
stains. The routinely used hematoxylin and eosin stain candida species poorly. The
specific fungal stains such as Gridley’s PAS stain and Gomori’s methenamine silver
are widely used for demonstrating fungi in the tissues.
Physiological Tests[17]
The main physiological tests used in definitive identification of Candida species
involve determination of their ability to assimilate and ferment individual carbon
and nitrogen sources and formation of germ tube in the presence of human serum or
better in egg white.
Phenotypic Methods[22]
Serotyping should prove useful for studying the epidemiology of candidiasis in hospitalized
patients.
Serotyping is limited to two serological groups (A and B), a fact that makes it inadequate
as an epidemiologic tool. It has recently been shown that there can be wide discrepancies
in results obtained with different methods of serotyping.
Resistogram Typing
This method is based on the differences in resistance of Candida isolates to six selected
organic and inorganic chemicals incorporated in agar medium. Thirteen resistogram
strains of C. albicans were found among isolates obtained from mouth. Resistogram do not correlate with
pathogenic potential, and even though improvement have been made in the method, growth
endpoints often present problems because of inoculum size, interpretation, and reproducibility.
Yeast Killer Toxin Typing
Owing to it reliability, economy, and versatility, this method can be used as an alternative
biotyping method in laboratories lacking the financial and training resources. Initially,
the method used nine killer strains, now it has expanded to using 30 killer strains
and 3 antifungal agents, which appeared to discriminate between sufficient number
of strains of C. albicans.[22]
[23]
Morphotyping
It is a simple and easy typing method using Sabouraud triphenyltetrazolium agar as
a tool for differentiation and morphotyping of Candida subspecies. This method has
been used in a study of morphotypes of 446 strains of C. albicans isolated from various clinical specimens.[24]
Biotyping
It is a simpler method. This system is comprised of three tests, the API ZYM system,
the AP20C system, and a plate test for resistance to boric acid. This system was found
to distinguish a possible of 234 biotypes which can be distinguished by one- or two-dimensional
gel electrophoresis. These methods have been used to separate C. albicans at the subspecies level.[25]
Genetic Methods
The earliest molecular methods used for fingerprinting C. albicans strains were karyotyping, restriction endonuclease analysis, and restriction fragment
length polymorphism. In arbitrarily primed polymerase chain reaction (PCR) analysis,
the genomic deoxyribonucleic acid is used as a template and amplified to a low annealing
temperature with the use of single short primer (9–10 bases) of an arbitrary sequence.
Multiplex PCR has been used to identify various candida species. Real-time PCR has
demonstrated that Candida biofilms can exert resistance to many commonly employed
antifungals in clinical setting.
Serological tests for detection of invasive candidiasis agar gel diffusion, whole
cell agglutination, and counterimmunoelectrophoresis are of lesser importance. Candida
enolase antigen or antibody detection are of importance for differentiating colonization
and oral candidiasis and they are found in biofilm also.[2]
Immunodiagnosis
The use of specific antibodies labeled with fluorescent stain permits causative organisms
to be diagnosed accurately within minutes. However, the preparation of specific antisera
and purified polyclonal or monoclonal antibodies entails a much more extensive technical
outlay, so the application of these reagents need only be considered when a very precise
diagnosis is of therapeutic consequence.
Management
Assessment of predisposing factor plays a crucial role in the management of candida
infection. Mostly, the infection is simply and effectively treated with topical application
of antifungal ointments. However, in chronic mucocutaneous candidiasis with immunosupression,
topical agents may not be effective. In such instances, systemic administration of
medications is required.
Treatment: When topical therapy does not show good result then start with systemic
therapy because failure of drug response is the initial sign of underlying systemic
disease. Follow-up after 3 to 7 days is important to check the effect of drugs. Always
continue the treatment for 2 weeks after resolution of the lesions. Main goals of
treatment are to identify and eliminate possible contributory factors, to prevent
systemic dissemination, to eliminate any associated discomfort, and to reduce load
of Candida.[4]
Primary Line of Treatment
Nystatin is the drug of choice as a primary line of treatment, and for the mild and
localized candidiasis other drugs includes clotrimazole which is available as lozenges
and amphotericin B as oral suspension.
Nystatin: It is available as cream and oral suspensions. It is to be applied four
times a day and allowed to act approximately for 2 minutes in the oral cavity and
then it is to be swallowed. Nystatin shows no significant drug interaction or side
effects.
Amphotericin B: Amphotericin B is available as lozenge (Fungilin 10 mg) and oral suspension
(100 mg/mL) which is to be applied three to four times daily. It inhibits the adhesion
of Candida to the epithelial cells. It is a nephrotoxic drug.
Clotrimozole: Clotrimozole reduces the fungal growth because this drug inhibits the
synthesis of ergosterol which is a part of cell membrane of fungi. It is not indicated
for systemic infection. This drug is available as creams and lozenges (10 mg).
Second line of treatment: The second line of treatment is used for severe, localized,
immune suppressed patients and patients who respond poorly to primary line of treatment.
Drugs mainly used in second line of treatment are ketoconazole, fluconazole, and itraconazole.
Fluconazole: It is used in oropharyngeal candidosis.
Itraconazole: It is one of the broad spectrum antifungal drugs. It is contraindicated
in pregnancy and liver disease.
Prognosis: The prognosis is good for oral candidiasis with appropriate and effective
treatment. Relapse, when it occurs, is more often than not due to poor compliance
with therapy, failure to remove and clean dentures appropriately, or inability to
resolve the underlying predisposing factors to the infection.
