Clinics in Dermatology (2010) 28, 178184

Candidosis, a new challenge

Rubn Lpez-Martnez, MD
Departamento de Microbiologa y Parasitologia, Facultad de Medicina, Universidad Nacional Autnoma de Mxico, Ciudad Universitaria, Ave. Universidad 3000, C.P. 04510, Mexico D.F., Mexico

Abstract Superficial candidosis is a common fungal infection that could become a gateway to systemic spread. Candida albicans is the most important Candida spp; recently, so-called emergent species, such as C dubliniensis, C famata, and C lipolytica have been isolated. This chapter describes the clinical manifestations and laboratory diagnostic techniques, including direct examination, smears, cultures, and physiologic tests. Topical antifungal drugs available for the treatment of superficial candidosis, including imidazoles, triazoles, allylamines, and nystatin, are also discussed. For granulomatous and invasive forms of candidosis, triazoles, allylamines (terbinafine), echinocandins (caspofungin), and amphotericin B are elective therapeutic choices. It is important to eliminate associated predisposing factors that contribute to infection and, if possible, all samples obtained should be evaluated for cases of resistance. 2010 Elsevier Inc. All rights reserved.

Introduction
Mycoses have recently shown an important increase in their incidence because the general population is more exposed to factors that favor mycosis infection. Many times, these predisposing factors are related to immunodeficiency. In the case of candidosis, these include treatment with antibiotics, steroids, cytostatic, and immunodepressant drugs; organ and bone marrow transplantation; diabetes; leukemia, lymphoma, and other types of cancer; AIDS; and malnutrition. Candidosis is the emerging mycosis that has the greatest effect due to its frequency and the severity of its complications. Superficial candidosis is one of the most common clinical forms. It is characteristically chronic and recurrent, and at times, indicates the beginning of severe forms of this mycosis.1 One of the problems in clinical practice is the over-diagnosis or under-diagnosis of this infection, which leads to therapeutic errors.

Laboratory diagnostic techniques for candidosis can frequently produce false-positive or false-negative results if the appropriate method is not applied. In many instances, when traces of Candida yeast are observed or isolated on the skin or in the secretions being analyzed, laboratories report a positive result without taking into consideration that this yeast is a normal element of the flora of the skin and mucous membranes. The treatment of this mycosis is another important challenge. Despite new and more effective antimycotic drugs, therapies often fail because of ignorance regarding doses and therapeutic regimes or because of its increasing resistance to antifungal drugs. Therefore, antifungal sensitivity studies should be undertaken, when possible, before antifungal therapy is started.

Etiologic agents
Of the approximately 200 species of the genus Candida, only 12 are principal agents of disease.2 They are oval, elliptical, or cylindrical unicellular or bicellular

E-mail addresses: rlm@servidor.unam.mx, rlmmex@hotmail.com. 0738-081X/$ see front matter 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.clindermatol.2009.12.014

Candidosis, a new challenge yeasts measuring 3 to 5 m, with two-layer cell walls. They can develop true hyphae or braided, branching pseudohyphae. Colonies in Sabouraud cultures are whitish and creamy and exhibit no carotenoid or melanic pigments. Most live freely and grow on various substrates rich in organic matter, such as foods, vegetables, and debris. Some of them are residents on the human body, living on the skin and various mucous membranes, including the mouth, vagina, urethra, digestive tract, and upper respiratory area. Colonization begins in the first days after birth and persists throughout life. Taxonomically, Candida belongs to the phylum Ascomycetes, class Blastomycetes, order Cryptococcales, family Cryptococcaceae, and genus Candida.3 In 1995, a new species was identified,4 C dubliniensis, which is closely related to C albicans both morphologically and physiologically but is differentiated from it by its lack of reactivity to the Ca3 probe of C albicans and its intracellular -glucosidase activity. A new species, C nivariensis, which is closely related to C glabrata was suggested in 2005 based on phenotypic and molecular phylogenetic studies.5 Numerous species of Candida have been reported to cause infection, and many of these are classified as emergent.6,7 The most common species and their perfect (sexual) stages are listed in Table 1.

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Cutaneous candidosis
The most frequent sites of infection are the bending creases or intertrigos, between the fingers or toes, under the breasts, under the gluteus, the armpits, and groin (Figures 1 and 2). The main predisposing factors are maceration, heat, humidity, and obesity. The most common signs and symptoms are eruption, secretions, burning, and at times, pain. There can also be slight itching and whitish spots on the affected creases. In newborns, diaper dermatitis is frequently associated with candidosis, which exacerbates this pathology and makes diagnosis and treatment difficult. A differential diagnosis should be made with cases of dermatitis, allergies, contact dermatitis, and various eczemas, including infections by dermatophytes.

Mucosal Candida infection

Oral thrush This is one of the most frequent clinical forms. It occurs at all ages, but the symptoms are more aggressive in premature infants, lactating mothers, and elderly people. The most common predisposing factors are antibiotic therapy, corticosteroid treatment, dental prostheses, cancer, radiation therapy, and AIDS.8 The clinical modalities are: 1. Acute and chronic pseudomembranous thrush. The acute form, which is most common in lactating mothers and the elderly, is characterized by whitish membranous spots on the tongue, palate, cheeks, and pharynx (Figure 3). There is an eruption and slight burning. The chronic form often spreads to the esophagus. 2. Atrophic thrush can be acute or chronic. The oral epithelia are thin, burning, shiny, and edematous. There is atrophy, eruption, and ulcerations on the lingual

Clinical forms
Superficial candidosis is classified as: (1) cutaneous, (2) mucosal (oral, vulvovaginal, or balanopreputial), (3) paronychial and onychial, or (4) chronic mucocutaneous and granulomatous.

Table 1 Species C C C C C C C C C C C C

Main species of Candida causing different clinical forms Oral X X X X X X X X Vaginal X X X X X X X Cutaneous X X X X X X X Ungual X X X X X X X X GI X X X X X X Systemic X X X X X X X X X Fungemia X X X X X X X X X X X

albicans glabrata tropicalis krusei (Issatchenkia orientalis) a guilliermondii (Pichia guilliermondii) a parapsilosis kefyr (Kluyveromyces fragilis) a dubliniensis b famata b (Debaryomyces shansenii) a lipolytica b (Yarrowia lipolytica) a norvegensis b (Pichia norvegensis) a lusitaniae b (Clavispora lusitaniae) a

GI, Gastrointestinal. a Perfect stage = sexual forms. b Emergent species.

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R. Lpez-Martnez

Fig. 3

Acute pseudomembranous thrush on the tongue.

differential diagnosis is established with cases of leukoplakia, saburral tongue, buccal aphtha, and bacterial infection.
Fig. 1 Cutaneous candidosis infection in interdigital spaces.

mucosal membrane. This form is frequently observed in people with dental prostheses. 3. Leukoplakia appears as localized whitish spots with irregular borders that are hard to remove. 4. Angular cheilitis affects the corners of the mouth and is accompanied by cracking of the skin and whitish scaling. Pain usually occurs when chewing (Figure 4). It commonly spreads to the skin in the perioral area. The main predisposing factors are malnutrition, excessive salivation (sialorrhea), and maceration of the lingual creases. Oral yeast infections in AIDS patients frequently spread to the esophagus and the rest of the digestive tract. The

Vulvovaginal This can occur at any age. Its evolution is chronic. The main clinical manifestations are leukorrhea, eruption on the vulva and vagina, pelvic heaviness, and moderate itching. It can spread to the perineum and the groin creases and is mainly observed in pregnant mothers, women with intrauterine devices, or women who use oral contraceptives. It is very frequently associated with diabetes mellitus, obesity, or corticotherapy. A differential diagnosis must be made in cases of trichomoniasis and bacterial vaginosis. Balanopreputial This occurs mainly in uncircumcised men and typically appears as papules, pustules, irritation, pain, and sometimes, whitish sores and secretions on the glans and prepuce (Figure 5). These are painful and recurrent. The differential diagnosis

Fig. 2 Cutaneous candidosis infection frequently occurs under folds of the breast.

Fig. 4 Angular cheilitis affects the corners of the mouth and is accompanied by cracking of the skin and whitish scaling.

Candidosis, a new challenge is established in cases of bacterial balanitis and genital herpes. The predisposing factors are a lack of cleanliness around the genitals, redundant prepuce, and other factors related to diabetes and immunodeficiency.

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Paronychial and onychomycotic candidosis

This entity is more common on the fingernails. It affects all ages and it is frequent in diabetic individuals. The sores are found mainly on the extreme proximal region of the fingernail, accompanied by greenish-yellow, ochre, or whitish discoloration. Generally, there is subungual oncolysis with separation of the extreme distal part of the fingernail. When it occurs on the dorsum of the nail plate, it is called superficial white onychomycosis. Paronychia frequently occurs, which usually develops with irritation, edema, and pain when pressure is applied (Figure 6). The predisposing factors are constant humidity, maceration, frequent manicures, and vascular diseases of the limbs. It must be differentiated from onychomycosis and from other onychopathies, such as leukonychia, trauma, psoriasis, and melanoma.

Fig. 6 Nails demonstrate paronychia, which usually develops with irritation, edema, and pain when pressure is applied.

usually resistant to available therapiesm and the most important risk in patients with altered T lymphocytes and deficiencies in -globulin is dissemination to deep tissues, which can lead to septicemia and death.

Chronic mucocutaneous candidosis and granuloma

This occurs infrequently, but when it does, it is usually very severe. It affects all the tissues of the skin. The most common locations are the face, skin, scalp, and hands. It is chronic and causes pain of the affected areas. Hyperkeratosis occurs with thickening of the skin and subcutaneous tissues, and pain. The lesions become hyperkeratotic with time. It is

Laboratory diagnosis
When cutaneous candidosis is present in immunocompromised patients, such as individuals with diabetes, leukemia, lymphoma, transplants, or cancer, the possibility of systemic candidosis must be entertained. The isolation and identification of Candida spp requires proper processing and interpretation of the diagnostic methods. The various yeasts must be isolated in samples of skin and mucous membranes where Candida is usually present. When the yeasts are initially isolated from deep tissues, the presence of only one variety of yeast has diagnostic value.

Direct microscopic examination

A drop of excreted material, such as blood, urine, or liquid from the whitish spots on the skin lesions is suspended between upper and lower slides. A drop of saline solution is added, and the sample is observed under a microscope for the presence of pseudomycelia and spores of the yeast (Figure 7).

Smears
To see yeasts and pseudohyphae better, it is useful to produce smears from solid specimens, such as biopsies or macerated tissue, and stain them with periodic-acid Schiff (PAS), Giemsa, Gram, or Gomori-Grocott.

Culture
Fig. 5 Balanopreputial Candida infection typically appears on the glans and prepuce.

The specimens are placed on Sabouraud dextrose agar (ss) and on Sabouraud agar with added chloramphenicol

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R. Lpez-Martnez more than 50% of the sample. To produce chlamydoconidia, a colony of yeast from a culture is plated on cornmeal agar and incubated for 2 days. Yeasts with pseudohyphae and double-walled chlamydoconidia are observed microscopically. Positive results in these two tests confirm the diagnosis of infection with these species. Chromogenic culture media, such as CHROMagar (CHROMagar Microbiology, Paris, France), are very useful for the diagnosis of Candida. In these cultures, some Candida spp produce colonies of different colors: C albicans is light green, C dubliniensis is bright green, and C parapsilosis is pale pink. For identification of other species, physiologic tests of carbohydrate assimilation are used, including auxanograms. Automated and semiautomated commercial kits are currently available, such as Api C-32 or Vitek (bioMrieux sa, Marcy l'Etoile, France) for these examinations.

Fig. 7 Photomicrograph shows the presence of pseudomycelia and yeasts ( 400).

and cycloheximide antibiotics (SA). The species C tropicalis, C krusei, and C parapsilosis are sensitive to the latter medium. All Candida spp grow on SalmonellaShigella agar and only some of them on Sabouraud agar, including C albicans, C dubliniensis, C kefyr, and C guilliermondii. Optimal growth of Candida spp occurs at room temperature, and colonies appear in 48 to 72 hours (Figure 8). To maximize the diagnostic value of a study, it is recommended that direct examinations, smears, and cultures be performed in triplicate.

Immunologic studies
Immunologic studies are not always necessary in cases of superficial candidosis; however, in deep infections, these tests are valuable for diagnosis and prognosis. Precipitation, agglutination, and complement fixation reactions are used to detect antibodies. To detect antigens, radioimmunoassays (RIAs) are used, and to detect antigens and antibodies, enzyme-linked immunosorbent assays (ELISAs) and DotELISAs are used. To assess definitive fungal metabolites, Darabinol and 3 glucans are determined.

Physiologic tests Molecular tests

Two tests are used to identify C albicans and C dubliniensis: filamentation in serum and the production of chlamydoconidia. In the first test, an aliquot of the culture is placed in human or rabbit serum (5 mL) and incubated at 37C for 4 hours. The test is positive if filaments are found in Molecular tests are mainly used in research but are very useful for verifying the results of routine studies, or in taxonomy, to separate the species previously considered a single species. As an example, C dubliniensis was separated from C albicans, and C nivariensis from C glabrata. Conversely, two or more different species have been joined into a single species based on the similarities of their DNA sequences. For instance, C claussenii, C stellatoidea, and C. langeroni correspond genetically to C albicans, C pseudotropicalis was merged into C kefyr, and C paratropicalis was merged into C. tropicalis. As a consequence, these species have disappeared from today's taxonomy.

Histopathology
As with immunologic analyses, histopathologic studies are not routinely used for the diagnosis of superficial candidosis; however, histopathology must occasionally be used in chronic mucocutaneous candidosis or in cases of clinical doubt. The most common staining techniques used for fungi are PAS, Gomori-Grocott, and hematoxylin and eosin (Figure 9).

Fig. 8 Optimal growth of Candida spp occurs at room temperature, and colonies appear in 48 to 72 hours.

Candidosis, a new challenge

183 200 mg every 12 hours). This triazole is more effective than fluconazole. Among the echinocandins, caspofungin is the drug of choice, because it inhibits -(1,3)-D-glucan synthase in the cell wall at a dose of 70 mg on the first day and thereafter at 50 mg daily for 30 days. Other echinocandins, such as anidulafungin and micafungin, are also effective.13-15 Micafungin is very effective against Candida spp that are resistant to other antifungals, including C glabrata, C krusei, and C lusitaniae.

Oral candidosis
One of the most commonly used drugs is nystatin as a solution, administered as a rinse 2 to 3 times a day. It is recommended that the rinse be swallowed. In serious and very extensive cases of oral candidosis, the aforementioned oral triazoles should be administered for cutaneous candidosis.

Fig. 9 Candida oesophagitis. Mycelial and yeastlike elements. Gomori's methenamine silver stain ( 1000).

Vulvovaginal candidosis

Treatment
The rule in the therapy of opportunistic mycoses is to eliminate the predisposing factors. This is not always possible with underlying diseases such as cancer, leukemia, lymphoma, AIDS, and diabetes. It is, nevertheless, usually feasible to control some factors, such as the administration of antibiotics, steroids, and immunosuppressants; humidity, local maceration, vaginal pH, and fitting dental prostheses. Numerous useful antifungal drugs are available for the treatment of superficial candidosis.9,10 The most commonly recommended will be discussed. In general, topical antifungal agents are effective. Systemic antifungal agents are used only when there is resistance or a poor response to topical treatments, for granulomatous or chronic mucocutaneous candidosis, and for candidosis complicated by other pathologies. Topical azoles in the form of vaginal creams or suppositories are commonly used. These include butoconazole, clotrimazole, econazole lipogel, fenticonazole, ketoconazole, miconazole, omoconazole, oxiconazole, and terconazole. Oral triazoles, such as fluconazole as a single dose of 150 to 300 mg, can also be given. In cases of balanopreputial candidosis, the same scheme of treatment with topical creams and oral antifungals are used as discussed for cutaneous candidosis.

Paronychia and onychia

Systemic treatments are used. These include itraconazole at 200 mg daily for 3 months, or 200 mg every 12 hours for one week each month for 3 months; fluconazole at 1 to 2 mg/ kg daily in children aged older than 1 year until cured and 150 to 300 mg weekly for 4 to 6 weeks in adults; and terbinafine at 250 mg daily for 3 months. Topical treatments can be used for lactating mothers and preschool children when less than 50% of the fingernails are affected or when the patient is intolerant to oral antifungal drugs. These include morpholines (amorolfine solution, 1 weekly application for 6 months), and hydroxypyridines (ciclopiroxolamine spray, once-daily application on alternate days for 1 month, then twice a week for 1 month, and once a week in the third month). Another alternative is voriconazole intravenously at a dose of 4 mg/kg every 12 hours or orally at 100 to 200 mg every 12 hours.16

Cutaneous candidosis
If the affected area is erythematous, only topical antifungal agents, such as imidazoles (eg, bifonazole, clotrimazole, fenticonazole, isoconazole, ketoconazole, miconazole, omoconazole, oxiconazole, terconazole) or allylamines (eg, terbinafine) are applied. These topical antifungals are applied twice daily until the patient is cured. If the lesions persist, ketoconazole should be given orally at 200 mg daily for 7 days. In more serious cases, systemic triazoles, such as fluconazole (150 to 300 mg weekly for 4 weeks) or itraconazole (200 mg every 12 hours for 4 weeks) are recommended. A new triazole, posaconazole, in oral suspension (800 mg daily for 3 weeks) is effective for most Candida spp. It also induces very low resistance in many pathogenic fungi.11,12 An alternative for the treatment of Candida is voriconazole intravenously (4 mg/kg every 12 hours) or orally (100 to

Mucocutaneous granulomatous candidosis

Systemic antifungal treatments are used for cutaneous candidosis. If there is a poor clinical response or resistance to these antifungal drugs, amphotericin B deoxycholate is administered intravenously at an initial dose of 0.1 mg/kg per

184 application every third day, with a progressive increase of the dose to 0.7 mg/kg per application. It is important to consider intolerance to the drug or toxicity (mainly nephrotoxicity). In such cases, the dose must be adjusted in patients with a reduced glomerular filtration rate. When candidosis are associated with invasive forms, the administration of next triazoles is recommended: itraconazole, 200 mg every 12 h for four weeks; fluconazole, 150 300 mg weekly for four weeks; voriconazole IV, 4 mg/kg every 12 h; posaconazole, 800 mg daily for 30 days. Another drug of choice is caspofungin at a dose of 70 mg on the first day and therafter at 50 mg/day/30 days. A new challenge in the treatment of mycoses is the development of resistance to antifungal agents, which has been observed mostly with fluconazole and itraconazole. Susceptibility testing to identify any potentially resistant Candida spp should be performed.

R. Lpez-Martnez
4. Sullivan DJ, Westerneng TJ, Haynes KA, et al. Candida dubliniensis sp. nov.: phenotypic and molecular characterization of a novel species associated with oral candidosis in HIV-infected individuals. Microbiology 1995;141:1507-21. 5. Alcoba-Flores J, Mndez-Alvarez S, Cano J, et al. Phenotypic and molecular characterization of Candida nivariensis sp. nov., a possible new opportunistic fungus. J Clin Microbiol 2005;43:4107-11. 6. Krcmery V, Barnes AJ. Non-albicans Candida spp. causing fungaemia: pathogenicity and antifungal resistance. J Hosp Infect 2002;50:246-60. 7. Agarwal S, Thakur K, Kanga A, et al. Catheter-related candidemia caused by Candida lipolytica in a child with tubercular meningitis. Indian J Pathol Microbiol 2008;51:298-300. 8. Samaranayake LP. Nuevas perspectivas en la epidemiologa y etiopatognesis de la candidiasis oral. Gac Med Bilbao 2001;98:E15-6. 9. Lpez-Martnez R. Frmacos tiles en el tratamiento de las micosis. In: Rodrguez-Carranza R, Vidrio-Lpez H, Campos Seplveda AE, editors. Gua de farmacologa y teraputica. 2nd ed. McGraw Hill: Mxico, DF; 2009. p. 276-81. 10. Chen S, Sorrell T. Antifungal agents. Med J Austral 2007;187:404-9. 11. Vzquez JA, Skiest DJ, Nieto L, et al. A multicenter randomized trial evaluating posaconazole versus fluconazole for the treatment of oropharyngeal candidiasis in subjects with HIV/AIDS. Clin Infect Dis 2006;42:1179-86. 12. Skiest DJ, Vzquez JA, Anstead GM, et al. Posaconazole for the treatment of azole-refractory oropharyngeal and esophageal candidiasis in subjects with HIV infection. Clin Infect Dis 2007;44:607-14. 13. Vzquez del Mercado E, Arenas R. Antimicticos en nios. Dermatol Peditr Lat 2007;5:155-64. 14. Bennet JE. Echinocandins for candidemia in adults without neutropenia. N Engl J Med 2006;355:1154-9. 15. Jarvis B, Figgitt DP, Scott LJ. Micafungin. Drug 2004;64:969-82. 16. Scott LJ, Simpson D. Voriconazole. A review of it use in the management of invasive fungal infection. Drugs 2007;67:269-98.

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