Oral Candidiasis- A review
Dr. Sameer1, Dr. Pooja Narain2, Dr. V N Jhameria3, Dr D K Gupta4, Dr Farzan Rahman5,
Dr. Juhi Narain6
- Assistant Professor, Oral & Maxillofacial Surgeon, Department of Oral & Maxillofacial Surgery, Government Dental College & Hospital, Jaipur, India.
- Senior Lecturer, Oral Pathologist, Department of Oral Pathology & Microbiology, Rajasthan Dental College & Hospital, Jaipur, India.
- Professor, Paediatric Surgeon, Department of Paediatric Surgery, J K Lon Hospital, Jaipur, India.
- Professor, Oral & Maxillofacial Surgeon, Department of Oral & Maxillofacial Surgery, Government Dental College & Hospital, Jaipur, India.
- Professor, Oral Pathologist, Department of Oral Pathology & Microbiology, Jaipur Dental College & Hospital, Jaipur, India.
- Dental Surgeon, Dental Clinic, Jaipur, India.
Abstract
Candidiasis, a common opportunistic fungal infection of the oral cavity, may be a cause of discomfort in dental patients. The article reviews common clinical types of candidiasis, its laboratory diagnosis current treatment modalities. The majority of infections are due to Candida albicans (C. albicans) although other species such as C. glabrata, C. tropicalis, C. krusei and C. parapsilosis are increasingly isolated. In fact, C. albicans may be a component of normal oral microflora, with as many as 30% to 50% of people simply carrying the organism in their mouths without clinical evidence of infection. The frequency of invasive fungal infections has increased over the last decade with the rise in at risk populations of patients.
Keywords: candidiasis, oral candidiasis, candida albicans.
Introduction
Infection with the yeast like fungal organism Candida albicans is termed as candidiasis.[1] The first known description of Candida infections, oral candidiasis (thrush) in two patients with other underlying diseases may be found in Hippocrate's "Epidemics" from the fourth century B.C.[2] “Thrush” one form of oral candidiasis is perhaps one of the earliest oral diseases documented. Derivation of this term is rather obscure, but according to the Oxford Dictionary, it probably originated from the old Danish Torsk which is synonymously used both for the disease and the bird of that name. Rosen von Rosenstein (1771) was the first to attempt to divide the disease into categories based on the severity and distribution of the lesions.[3] The fungus now known as Candida albicans was isolated by Bennett (1844) from the sputum of a tuberculosis patient, by Wilkinson (1849) from vaginal candidiasis. Robin (1853) was the first to observe concomitant thickening of the epithelium in lesions resembling thrush.[4]
Classification
The taxonomic position and classification of the thrush fungus was the subject of much debate and controversy for several decades as a result of different morphologic forms of the organism and different synonyms for the same fungus.[5] Robin (1853) had named it Oidium albicans, Quinquaud (1868) syringospora robinii and Reess (1875) Saccharomyces albicans. The first binomial to gain wide acceptance over a long period and which sometimes is still albeit wrongly used was Monilia albicans, which was suggested by Zopf in 1890. Berkhout in 1923, after recognizing the differences between Monilia species isolated from rotting plants and fruits and the yeasts isolated from medical cases, established the genus Candida to accommodate the latter. This was accepted as the official name of the genus by the Eighth Botonomical Congress in Paris in 1954.[4] Berkout5 proposed the name Candida from the Latin toga candida, which referred to the white robe worn by candidates for the Roman senate. Albicans also comes from the Latin albicare which means "to whiten". This translation corresponds well with what is considered traditionally and reported clinically as a presentation of candidal mucosal infection characterised as a white milk curd like covering of the mucosa, which wipes off easily.[6] The Index Medicus has not recognized the genus Monilia or the term Moniliasis in reference to human disease since 1981.[5] There have been around 166 synonyms being recognized for Candida albicans worldwide.7 The genus Candida is within the class Deuteromycetes and has been described as a "taxonomic pit" into which yeasts without a known sexual stage or other remarkable phenotype character have been thrown. Its members are biologically diverse and include yeasts with ascomycetous and basidiomycetous affinities. There are currently between 150 and 200 species recognized in the genus.[8] The genus Candida includes characteristically white asporogenous (imperfect) yeasts capable of forming pseudohyphae. Within the genus, species are characterized primarily by colonial morphology, carbon utilization, and fermentation.[9] There are seven Candida species of major medical importance, the most important being Candida albicans, the one most frequently isolated. It is believed to be the most virulent in man and can be isolated from human body as a commensal or as an opportunistic pathogen.[10]
The other Candida species encountered in human infections are C. tropicalis, C. glabrata, C. parapsilosis, C. stellatoidea, C. pseudotropicalis, C. guillermondii, C. krusei, and C. kyfer. These species are usually regarded as opportunists.[10,11]
CLINICAL SPECTRUM
The oral lesions of candidiasis have a different appearance and occur in several clinical forms. Several attempts have been made to classify these forms.[12] By tradition the most frequently adopted classification has been the one proposed by Lehner (1966)[13] who classified it based on clinical, mycological, histological, serological and therapeutic criteria. He divided oral candidiasis into acute and chronic types and further subdivided each of the latter as follows:
Acute:
Acute Pseudomembranous Oral Candidiasis. (Thrush)
Acute Atrophic Oral Candidiasis
Chronic:
Chronic Hyperplastic Oral Candidiasis
Chronic Atrophic Oral Candidiasis
Chronic hyperplastic candidiasis was further subdivided into 4 groups based on localization pattern and endocrine involvement as follows:
1. Chronic oral candidosis (candidal leukoplakia)
2. Endocrine candidosis syndrome
3. Chronic localized mucocutaneous candidiasis
4. Chronic diffuse candidosis
Chronic atrophic candidosis includes denture sore mouth and angular cheilitis caused by Candida. Lehner has suggested the following diagnostic criteria of oral candidosis
1. White plaques or diffuse erythematous areas
2. Culture of Candida from the saliva
3. Presence of the mycelium on direct examination of a smear from the lesion
4. Biopsy examination showing hyphae in the epithelium
5. Characteristic histologic changes and
6. Serum fluorescent antibody titre against Candida albicans greater than 1:16 and a positive antibody test with neat saliva.
Samaranayake and Yaccob[14] noted that the subdivision of chronic hyperplastic candidosis generates confusion particularly because it lumps together candidosis that are localized in the oral cavity alone and oral manifestations of mucocutaneous candidosis. It was therefore suggested that oral candidiasis should have dichotomatous classification, delineating primary oral candidiasis, that is infections exclusively confined to oral and perioral tissues (category 1) from secondary oral candidosis which are present in cutaneous as well as mucosal surfaces of the body. (Table 1)
Holmstrup and Besserman (1983)[15] noted that Pseudomembranous candidiasis is not always acute but may last for many months in certain categories of patients (such as AIDS patients). Also the value of using the term atrophic to describe erythematous areas is limited since the redness of the oral mucosa may be caused by increased vascularity with or without reduced thickness of epithelium.
Holmstrup and Besserman suggested future classification be based on clinical and histopathological terms like erythematous, plaque like and nodular lesions. (Table 2)
They proposed that a future revised clinical classification of primary oral
candidosis should comprise the following entities as shown in the above table.
Furthermore, in 1990 Holmstrup and T Axell[16] made an attempt to reclassify oral candidiasis mainly based on clinical terms. (Table 3)
Recently, T Axell, Samaranayaka and Reichart (1997)[17], have proposed a reclassification of oral candidiasis first because according to them there has been an almost indiscriminate application of the nomenclature related to the clinical subdivision of oral candidiasis and second because unusual clinical variants have appeared with the pandemic progression of the HIV infection.
There are great clinical similarities between the plaque-like and nodular lesions and different forms of oral leukoplakias. The latter are often super infected with Candida and are then called candidal leukoplakias. Previous classifications do not clearly take into consideration superinfection of other lesions with Candida. Such lesions are often keratinized.
To minimize diagnostic confusion they have introduced and added another entity to the clinical classification of oral candidiasis - keratinized primary lesions super infected with Candida. This entity would comprise leukoplakia, lichen planus and lupus erythematosus. Leukoplakias are infected with Candida in about 10% of the cases, lichen planus in 40 % and lupus erythematosus in 50%.
Cheilocandidosis described by Reade et al (1982)[18] and Juvenile Juxtavermillion Candidosis discovered by Bouquot and Fenton (1988)[19] are newer clinical entities, which manifest around the lips of affected individuals and respond well to antifungal therapy; are thought to be due to either primary or secondary infection by Candida species. Samaranayake and Yaacob (1990)[14] suggested that further clinical and microbiological evidence is required to justify their inclusion in a standard classification of oral candidiasis. Diagnosis of oral yeast infections should be based on a combination of laboratory methods as well as clinical signs and symptoms.[20] Oral candidiasis is divided into primary and secondary infections (Table 5). The primary infections are restricted to the oral and perioral sites, whereas secondary infections are accompanied by systemic mucocutenous infections.[21]
Methods
LABORATORY DIAGNOSIS OF ORAL CANDIDIASIS
A correct diagnosis provides the specific treatment of a fungal infection and may prove life saving or stave off the complications produce there in.[22] Olsen and Stenderup, (1990)[20] stated laboratory findings should be correlated with the severity of clinical signs and symptoms. Effectiveness of anti fungal medication also aids in diagnosis.[23]
The following are the guidelines for specimen collection.[23]
1. The specimen should be collected from an active lesion; old 'burned out' lesions often do not contain viable organisms.
2. Collect the specimen under aseptic conditions.
3. Collect sufficient specimen.
4. Use sterile collection devices and containers
5. Label the specimen appropriately; all clinical specimens should be considered as potential biohazards and should be handled with care using universal precautions.
The specimen should be kept moist or in a transport medium and stored in a refrigerator at 4ºC.[20] If sampled patients have infectious diseases such as tuberculosis, hepatitis, AIDS, etc., the diagnostic microbial laboratory must be informed about it so that specimens can be processed with caution and without risks of infection to the technical staff. Due to variety of clinical forms of oral candidiasis a number of different types of specimens may be submitted to the laboratory.[20]
1. Smear: Smears are taken from the infected oral mucosa, rhagades and the fitting side of the denture, preferably with wooden spatulas. Fixed immediately in ether/alcohol 1:1 or with spray fix. Dry preparations may be examined by Gram stain method and Periodic Acid Schiff (PAS) method.
2. Swabs: Swabs are seeded on Sabouraud’s agar (25ºC or room temperature), on blood agar (35ºC), on Pagano-Levin medium (35ºC) or on Littmann’s substrate (25ºC). Incubation at 25ºC is done to ensure recovery of species growing badly at 35ºC. Sabouraud’s dextrose agar is frequently used as a primary culture medium. Since mixed yeast infections are seen in the oral cavity more frequently than previously thought, particularly in immunocompromised or debilitated patients, Pagano-Levin agar or Littmann’s substrate, are useful supplements, because they enable distinction of yeasts on the basis of difference in colony color.
3. Biopsy: Biopsy specimen (either excisional or incisional) should in addition be sent for histopathological examination when chronic hyperplastic candidosis is suspected.
4. Imprint Culture Technique: Sterile, square (2.2 x 2.5 cm), plastic foam pads are dipped in peptone water and placed on the restricted area under study for 30 – 60 seconds. Thereafter the pad is placed directly on Pagano-Levin or Sabouraud's agar, left in situ for the first 8 hours of 48 hours incubation at 37ºC. Then, the candidal density at each site is determined by a Gallenkamp colony counter and expressed as colony forming units per mm2 (CFUmm-2).[20,24] Thus it yields yeasts per unit mucosal surface.[25] 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 (Budtz-Jorgensen, 1978, Olsen and Stenderup A, 1990).[20,26]
5. Impression Culture Technique: It involves taking maxillary and mandibular alginate impressions, transporting them to the laboratory and casting 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 48-72 hours at 37ºC and the CFU of yeasts estimated.
6. Saliva: This simple technique involves requesting the patient to expectorate 2ml 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.[27] Patients who display clinical signs of oral candidiasis usually have more than 400 CFU/mL.[28]
7. Oral Rinse Technique: It was first described by Mckendrik, Wilson and Main (1967) and later modified by Samaranayake et al (1968).[29]
8. Paper Points: An absorbable sterile point is inserted to the depth of the pocket and kept there for 10 sec and then the points are transferred to a 2ml vial containing Moller's VMGA III transport medium, (which also facilitates survival of facultative and anaerobic bacteria).[20]
9. Commercial Identification Kits: The Microstix-candida (MC) system consists of a plastic strip to which is affixed a dry culture area (10 mm x 10 mm) of modified Nickerson medium (Nickerson, 1953) and a plastic pouch for incubation. The O Yeast-I dent system is based on the use of chromogenic substances to measure enzyme activities. ricult-N dip slide technique is similar to, but of higher sensitivity than M-C system. Yeast-I dent system is based on the use of chromogenic substances to measure enzyme activities.[20]
Histological Identification of Candida species in Biopsy Specimens
Demonstration of fungi in biopsy specimens may require several serial sections to be cut.[20] Because of their size morphological diversity and polysaccharide content, fungi can be easily demonstrated and studied in tissue sections with special stains. The routinely used Haematoxylin and Eosin stain poorly stains Candida species. Hence they may be overlooked in tissue sections. The specific fungal stains such as Periodic Acid Schiff stain (PAS), Grocott-Gomori's Methenamine Silver (GMS) and Gridley stains are widely used for demonstrating fungi in the tissues, which are coloured intensely with these stains.[24] Calcoflour White Stain30 staining can be done in formalin fixed paraffin embedded specimens but a fluorescence microscope is required for visualization.
Physiological tests
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. These should be supplemented with morphological test observations such as germ tube formation for a reliable identification of Candida species.[24]
A) Carbohydrate Assimilation Test[31]
Carbohydrate utilization tests are the most widely used methods for the definitive identification of clinically important yeasts. (Sandven P, 1990)[31]
B) Nitrate Assimilation
Methods used are the same as for carbon assimilation tests, but in these tests basal media must be nitrogen free and must include glucose as a carbon source.
C) Carbohydrate Fermentation
Carbohydrate fermentation tests are useful tests for differentiating species and can be used to supplement carbohydrate utilization test results, for differentiating species and thus making a definitive identification of an organism. (Sandven P 1990)[31]
The assimilation reactions and fermentation reactions of Candida species are tabulated in Table 6 and Table 7.
Note: Glu = Glucose, Mal = Maltose Suc = Sucrose Lac = Lactose Cel = Cellobiose, Gal = Galactose, Tre = Trehalose, Raf = Raffinose, Mel = Melibiose, Xyl = Xylose, Ino = Inositol, Dul = Dulcitol; + = Positive reaction, -- = Negative reaction,
Note: Glu = Glucose, Mal = Maltose Suc = Sucrose Lac = Lactose
+ = Positive reaction, -- = Negative reaction, A= Acid Production G = Gas Production.
STRAIN DIFFERENTIATION OF CANDIDA ALBICANS[32]
As Candida albicans is both a commensal and a pathogen, there is every need to identify the pathogenic strain and the virulence factor in it so that the diagnosis of carrier state and infection becomes clearly delineated. Ideally, a method for differentiating Candida albicans at a subspecies level should be able to discriminate between strains which are epidemiologically unrelated & be highly reproducible both intra, and inter laboratory, take minimal time, be capable of processing a large number of strains, require a minimum of specialized equipment, and be relatively inexpensive.
Phenotypic methods
1. Serotyping: Serotyping is limited to the two serotypes (A and B), a fact that makes it inadequate as an epidemiologic tool. Furthermore, it has recently been shown that there can be wide discrepancies in the results obtained with different methods of serotyping, making it difficult to compare results obtained with different methods of serotyping.
2. Resistogram typing: It has been shown that resistograms do not correlate with pathogenic potential, and even though improvements have been made in the method growth end-points often present problems because of inoculum size, interpretation, and reproducibility.
3. Yeast ‘Killer Toxin’ typing: These authors initially used nine killer strains, developing a triplet code to distinguish between 100 strains of C albicans, and found 25 killer- sensitive types. This method was expanded by using 30 killer strains and three antifungal agents, which appeared to discriminate between sufficient numbers of strains of C. albicans.
4. Morphotyping:This method has been used in a study of the morphotypes of 446 strains of C.albicans isolated from various clinical specimens.[33]
5. Biotyping:They utilized proteinase and lipase production to distinguish four biotypes. Williamson (1987)[34] has proposed a simpler method. This system comprised three tests, the APIZYM system, the API 20C system, and a plate test for resistance to boric acid. This system was found to distinguish a possible 234 biotypes of which 33 were found among the 1430 isolates of C. albicans taken from oral, genital and skin sites.
6.5Protein typing:Non-lethal mutations of proteins during the yeast cell cycle yield proteins of differing physical properties between strains, which may be distinguishable by one or two dimensional gel electrophoresis. These methods have been used to separate C. albicans at the subspecies level. Anti C.albicans antiserum can be used for further distinction of protein differences between strains and this may be a useful epidemiologic method. Furthermore, specific isoenzyme differences between strains may be visualized by this method to provide a range of variation sufficient for epidemiologic study.
GENETIC METHODS
The earliest molecular methods used for fingerprinting C. albicans strains were karyotyping, restriction endonuclease analysis (REA), and restriction fragment length polymorphism (RFLP). To date, the majority of studies have focused on studying C albicans isolates from HIV infected patients. In arbitrarily primed polymerase chain reaction (AP- PCR) analysis (synonym: randomly amplified polymorphic DNA (RAPD) analysis), the genomic DNA is used as a template and amplified at a low annealing temperature with use of a single short primer (9 to 10 bases) of an arbitrary sequence. However, the reproducibility of AP-PCR is dependent upon the careful standardization of the PCR conditions, and the discriminatory power is dependent on the primer used and optimization of the assay (Williams et al, 1990;Dassanayake & Samaranayake, 2000).[31]
Serological Tests[32]:
The serological tests are useful for systemic fungal infections and have both diagnostic and prognostic values. (Table 8)
Immunodiagnosis[24]:
The use of specific antibodies labelled 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 (Olsen and Stenderup, 1990). The usefulness of antibody testing in the diagnosis of oral candidosis when other simpler, sensitive and reliable techniques are available is questionable (Silverman et al, 1990)[24]
THERAPEUTIC ASPECTS[35]:
For the normal healthy patient, the treatment of oral candidiasis is relatively simple and effective. Typically, topical medications are adequate. (Table 9) & (Table 10)
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