Inhibition of the growth in vitro of Trichophyton mentagrophytes, Trichophyton erinacei and Microsporum persicolor by miconazole and chlorhexidine

An agar dilution technique was used to assess the minimum inhibitory concentrations (MICs) of miconazole, chlorhexidine and a 1:1 combination of both agents for 9 isolates of Trichophyton mentagrophytes, 9 isolates of Trichophyton erinacei and 5 isolates of Microsporum persicolor. MICs of chlorhexidine did not vary significantly between the three dermatophyte species tested, but the MICs of miconazole alone and in combination with chlorhexidine for T. erinacei were significantly greater that those for T. mentagrophytes and M. persicolor. A synergistic drug interaction was noted with one isolate of T. erinacei and one isolate of M. persicolor. An additive effect was demonstrated for 13 isolates (5 T. mentagrophytes, 6 T. erinacei, 2 M. persicolor), and indifference was noted in 8 isolates (4 T. mentagrophytes, 2 T. erinacei, 2 M. persicolor). Although synergy was less often seen when compared with a previous study of Microsporum canis, the synergistic or additive effects seen with the majority (15 out of 23) of isolates studied in vitro provides a rationale for the combined use of miconazole and chlorhexidine in the adjunctive topical therapy of dermatophytosis caused by T. mentagrophytes, T. erinacei and M. persicolor.     

Dermatophytosis due to Microsporum persicolor: a retrospective study of 16 cases

A retrospective study of 16 cases of dermatophytosis due to Microsporum persicolor in dogs is reported. Hunting dogs were overrepresented (12/16). Skin lesions were observed on the face in all cases, but also on other locations (limbs, neck). The lesions included alopecia (15/16), erythema (13/16), scales (14/16), and crusts (13/16). Histopathology was performed in 10 cases and showed folliculitis and a lichenoid interface dermatitis. Fungal culture was positive in all cases and clinical resolution was achieved with standard antifungal agents (enilconazole, ketoconazole, griseofulvin). Two recurrences were observed (new contacts with rodents).     

Chronic dermatophytosis due to Microsporum persicolor infection in three dogs

Microsporum persicolor, a rare zoophilic dermatophyte, was isolated from three dogs with skin disease of between three and five years duration. Skin lesions consisted of scaling with minimal alopecia or erythema. Severe inflammatory changes were not observed clinically and pruritus was absent or mild. The face was affected in all three cases and more widespread lesions were found in two. The diagnosis of dermatophytosis was confirmed in each case by the demonstration of fungal hyphae in the epidermal stratum corneum on examination of skin biopsies. However, hair shaft invasion was not observed in either skin scrapings or histological sections. Of the three dogs, one partially improved following repeated courses of treatment, a second completely recovered with 11 weeks of combined topical and systemic therapy. Response to therapy could not be assessed in the remaining case.     

Canine nodular dermatophytosis (kerion): 23 cases

Dermatophytosis is a common zoonotic disease, and one of its clinical presentations in the dog is nodular dermatophytosis (kerion). Because the infection is located within the dermis, routine diagnostic tests such as a Wood's lamp examination, microscopic examination of hair shafts for fungal elements and fungal culture can yield negative results. In such cases, histopathological examination with routine and special stains (periodic acid-Schiff, Gomori methenamine silver) is required to confirm the diagnosis. Nodular dermatophytosis in 23 dogs of different breed, age and sex with single or multiple nodules is described. Twelve dogs had a single nodule, and 11 dogs showed multiple lesions. Wood's lamp examination was negative in all cases. Microscopic examination of plucked hairs showed arthrospores in 8 of 23 cases. Skin scrapings in mineral oil looking for arthrospores and/or hyphae were positive in 12 cases. Impression smears of exudates were diagnostic in 21 of 23 cases (91%), showing arthrospores within fragments of hair shafts or free among neutrophils and macrophages (pyogranulomatous inflammation). Histopathology was performed in two cases. Fungal culture was positive for Microsporum canis in 16 dogs and for Microsporum gypseum in one dog. In six cases, the causative agent was not identified by fungal culture. All dogs were treated with systemic antifungal therapy and in eight cases with concurrent antibiotic therapy. Nodular dermatophytosis resolved in all dogs with the prescribed treatments within 4 to 8 weeks. Transmission to people or other pets in the home was not found.     

Efficacy of pre-treatment with lufenuron for the prevention of Microsporum canis infection in a feline direct topical challenge model

Oral lufenuron is reportedly an effective treatment for some cats with dermatophytosis. The purpose of this study was to determine if lufenuron, when used as a pre-treatment prior to challenge exposure, would be protective against the development of infection after the direct topical application of fungal macrocondia (Microsporum canis spores). Three groups (n = 6/group) of juvenile cats were treated with either monthly oral lufenuron (30 or 133 mg/kg) or placebo. After 2 months of treatment, kittens were challenged using 10(5)Microsporum canis spores applied to the skin under occlusion. Cats were examined weekly and the following data collected: Wood's lamp examination; scoring for scale/crust, erythema and induration; lesion size; and the development of satellite lesions. Fungal cultures were performed bi-weekly. All cats became infected; the infections progressed, and then regressed, in a similar fashion in all groups. There were no consistent statistically significant differences in weekly infection scores between treated and untreated cats throughout the study. Treated cats did not recover faster than untreated cats. We conclude that oral lufenuron at the dosing schedule and conditions used in this study did not prevent dermatophytosis or alter the course of infection by direct topical challenge.     

Generalized Microsporum canis dermatophytosis in six Yorkshire terrier dogs

Six Yorkshire terrier dogs with generalized, chronic dermatophytosis caused by Microsporum canis were seen over a 3-year period. Specific tests showed that they also had concurrent leishmaniosis (four cases), leishmaniosis and ehrlichiosis (one case) or diabetes mellitus (one case). Although specific therapy for these infectious diseases was instituted and the dogs were treated systemically and topically with appropriate antifungal drugs, only partial clinical resolution of the dermatophytosis was achieved. M. canis infection resolved in the dog with diabetes mellitus after stabilizing the diabetes mellitus. Although immunological studies were not performed in these cases, it is theorized that the immune disregulation caused by leishmaniosis, ehrlichiosis or diabetes mellitus may have favoured generalization of the infection and prevented favourable responses to appropriate treatment of the M. canis infection.     

Dermatophytosis in red pandas (Ailurus fulgens fulgens): a review of 14 cases.

Fourteen cases of dermatophytosis were identified from medical records of red pandas (Ailurus fulgens fulgens) housed at the Knoxville Zoo between 1980 and 1996. The median age of affected animals on initial presentation was 8.5 wk (3 wk-11 mo). Clinical signs included crusting, purulent exudate, alopecia, thickening of affected skin, ulceration, and necrosis. Seven animals had mild lesions with signs restricted to crusting and/or alopecia, and six animals had more severe infections, with ulceration, skin necrosis, and purulent exudate. Five of the severely affected pandas had tail involvement. The severity of disease affecting one individual was not recorded. Dermatophytosis was confirmed by culture, cytology, histopathology, or culture followed by histopathology. Microsporum gypseum was the only fungal organism cultured. Six animals were treated for mild disease, and all clinical signs resolved. Partial tail amputation was required as part of the treatment regimen for two of the six severely affected animals, and two others had ulcerated tail lesions that left circumferential scarring after resolution of infection. Itraconazole (5 mg/kg p.o. q 12-24 hr) was the most frequently used systemic antifungal agent in animals with severe lesions. All fungal infections resolved, although one panda died from unrelated causes early in the treatment period.     

Dermatophyte infections in free-ranging Florida panthers (Felis concolor coryi).

Three free-ranging Florida panthers (Felis concolor coryi) were diagnosed with clinical dermatophytosis; two were infected with Trichophyton mentagrophytes, and one was infected with Microsporum gypseum. Two of these panthers were juvenile males that were diagnosed with focal to focally coalescing dermatophytosis; one caused by M. gypseum and the other by T. mentagrophytes. These animals were not treated, and clinical signs resolved spontaneously over 6 mo. The third panther, an adult male from southern Florida, presented with a diffuse dermatophytosis due to T. mentagrophytes infection. Initially, the panther had alopecia, excoriations, ulcerations, and multifocal pyoderma of the head, ears, neck, rear limbs, and abdominal region that progressed to lichenification of the skin and loss of nails from two digits. When topical therapy applied in the field at 45-day intervals was ineffective in clearing the infection, the animal was placed in captivity for intensive oral therapy to prevent further development of dermal mycosis, loss of additional nails, and spread of infection to other panthers. The panther was treated orally with itraconazole (9.5 mg/ kg) in the food s.i.d. for 6 wk. After treatment, nail regrowth occurred but the multifocal areas of alopecia remained. The panther was released back into the wild after two skin biopsy cultures were negative for fungal growth. Temporary removal of a free-ranging animal of an endangered species from its habitat for systemic treatment of dermatophytosis requires consideration of factors such as age, reproductive potential, holding facilities, treatment regimen, and the potential for successful reintroduction of the animal.     

The Value of Enzyme-linked Immunosorbent Assay (ELISA) in the Sero-diagnosis of Canine Dermatophytosis Due to Microsporum Canis

Abstract— Fourteen sera from dogs naturally or experimentally infected with Microsporum canis were examined by enzyme-linked immunosorbent assay (ELISA) in order to study the humoral response to infection. A group of 12 animals (7 laboratory dogs and 5 dogs from the natural environment) was used as controls. ELISA optical density (OD) values in twofold serial dilutions from 1:100 to 1:51200 were compared between the groups. Significant antibody response to infection with M. canis was observed in naturally and experimentally infected animals and confirmed by threshold determination. Specificity for the dermatophyte fungi was tested by performing cross-reactivity assays with three fungal antigens, Malassezia pachydermatis, Candida albicans and Aspergillus fumigatus. There were no statistically significant indications of cross-reactions, except with anti-A. fumigatus IgG, where differences were noticed within the groups of naturally infected animals and controls. The use of ELISA technique in sero-diagnosis of dermatophytosis in dogs is discussed. Résumé— 14 sérums chiens infectés naturellement ou expérimentalement par Microsporum canis ont été testés par une technique Immunoenzymatique (ELISA) pour étudier la réponse immunitaire humorale à cette infection. Le groupe témoin était composé de 12 animaux (7 chiens de laboratoire et 5 chiens d'un milieu naturel). Les valeurs de densité optique (DO) obtenues avec des dilutions sériées de raison 2 des sérums (1:100 à 1:51,500) ont été comparées entre chaque groupe. Des taux significativement élevés d'anticorps spécifiques de Microsporum canis ont été retrouvés chez les animaux infectés naturellement et expérimentalement et confirmés par l'établissement d'un seull de positivité. La spécificité de la technique vis-à-vis des dermatophytes a été appréciée en effectuant des tests d'antigénicité croisée avec des Malassezia pachydermatis, Candida albicans et Aspergillus fumigatus. II n'y avait pas de réactions croisées significatives, exception faite des IgG spécifiques d'A. fumigatus, pour lequel il existait des differences de réponse entre les animaux infectés naturellement et le groups témoin. L'utilisation d'une technique ELISA dans le diagnostic des dermatophyties canines est discutées. Zusammenfassung— Vierzehn Seren von Hunden, die natürlich oder experimentell mit Microsporum canis infiziert waren, wurde mit dem ELISA-Verfahren (Enzyme Linked Immunosorbent Assay) untersucht, um die humorale Antwort auf die lnfektion zu verfolgen. Eine Gruppe von 12 Tieren (7 Laborhunde und 5 Hunde aus normaler Haltung) dienten als Kontrolle. ELISA-optische Dichtewerte (OD) in zweifacher serieller Verdünnung von 1:100 bis 1:51200 wurden zwischen beiden Gruppen verglichen. Bei den natürlich und experimentell infizierten Tieren konnte eine signifikante Antikörperreaktion auf die lnfektion mit M. canis beobachtet und durch Schwellenwertbestimmung bestätigt werden. Die Spezifität für Dermatophyten wurde überprüft, indem Kreuzreaktionsproben mit drei Pilzantigenen, Malassezia pachydermatis, Canidida albicans und Aspergillus fumigatus, durchgeführt wurden. Statistisch signifikante Anzeichen für Kreuzreaktionen konnten nicht nachgewiesen werden, außer bei Anti-A. fumigatus-IgG, bei dem Unterschiede innerhalb der Gruppe der natürlich infizierten und der Kontrolltiere beobachtet wurden. Es wird über die Brauchbarkeit der ELISA-Technik für die Serodiagnose von Dermatophyten bei Hunden diskutiert. Resumen Se examinaron un total de 14 sueros procedentes de perros infectados de forma natural y artificial con Microsporum canis por el método de enzima ligado de inmunoabsorbencia, (ELISA), con el fin de estudiar la respuesta humoral a la infección. Como controles, se usaron un grupo de 12 animales (7 perros de laboratorio y 5 perros que vivían en condiciones naturales). Los valores de densidad óptica ELISA, (OD), fueron comparados entre los grupos en dos diluciones seriadas de 1:100 a 1:51,200. Una respuesta significativa de anticuerpos a la infección con Microsporum canis fue observada en perros infectados de forma natural y artificial, y confirmada por la determinación del punto crítico. La especificidad del dermatofito füngico se estudió por medio de ensayos de reactividad cruzada con otros tres antigenos fúngicos, Malassezia pachydermatis, Candida albicans y Aspergillus fumigatus. No se encontraron indicaciones de reacciones cruzadas estadísticamente significativas, excepto con la IgG de A. fumigatus, donde se advirtieron diferencias en el group de animales infectados naturalmente y en el grupo control. El uso de la téchnica ELISA en el diagnóstico serólogico de las dermatofilosis en el perro, es discutido.     

The Immune Response to Microsporum canis Induced by a Fungal Cell Wall Vaccine*

Abstract— An experimental infection model was used to assess induction of specific immunity against Microsporum canis in cats with an M. canis cell wall vaccine preparation. Kittens 8–9 weeks old (n= 12) received five doses of either vaccine or placebo at biweekly intervals. Specific immunity was monitored via plasma anti-dermatophyte antibody titers and lymphocyte blastogenesis (LB) to dermatophyte antigens. After vaccination, cats were challenged with viable M. canis spores, and lesion development was monitored. Vaccinated cats developed higher anti-dermatophyte IgG, but not IgM, titers than controls, beginning after the second dose of vaccine (P < 0.001). During the vaccination period, specific cellular immunity as measured by LB was absent in control cats, but developed to a limited degree in vaccinated cats (P < 0.05). After challenge with 10⁵ fungal spores per cat, both control and vaccinated cats developed active infections. The vaccine appeared to induce an antibody titer quantitatively similar to that produced by infection, but less measured cellular immunity than was seen with infection and recovery. These results suggest that induction of high titers of serum IgG or IgM antibody against Microsporum canis is not protective against challenge exposure.     

家兔皮癣病原菌的分离鉴定及致病性研究

从发病家兔的皮屑、被毛分离到两种病原真菌,通过培养特性、菌落性状、菌丝形态、孢子形状特征等一系列鉴定,确定为絮状表皮癣菌和石膏样小孢子菌.该菌通过皮肤涂擦法感染家兔,4天后均发病,产生以面部、头顶等处出现丘疹、瘙痒,继而皮损、流微黄色渗出液,日久结痂或鳞屑,逐渐消瘦为主要特征的一种皮癣病,与自然病例症状相同.     

Development of an enzyme-linked immunosorbant assay (ELISA) for the serodiagnosis of canine dermatophytosis caused by Microsporum canis

Abstract In dogs, dermatophytosis should be considered in any case of alopecic, papular or pustular lesion. The aim of this study was to develop an enzyme-linked immunosorbant assay (ELISA) as an aid in the diagnosis of canine dermatophytosis. The antigen used was a whole fungal extract obtained from an isolate of Microsporum canis cultured on a liquid medium from the parasitized hair of a cat with patches of alopecia. To assess the ELISA performances, sera from 18 dogs with dermatophytosis caused by M. canis (group A, n = 18), 20 dogs with skin diseases other than dermatophytosis and 22 healthy dogs (group B, n = 42) were tested. Four further animals were tested: three with dermatophytosis caused by M. gypseum and one by T. mentagrophytes. A significant difference (P < 0.01, Wilcoxon's test, w = 364) was found between IgG-specific levels of sera of recently M. canis-infected dogs (infection < 15 days) and controls (although three dogs had negative titres at this stage). A highly significant difference (P < 0.001, w = 462) was noted between controls and dogs with infection of longer duration (> 30 days). All dogs had positive titres at this stage. A highly significant correlation (P < 0.001, Spearman's test, rho = 0.86) between duration of infection and IgG concentration was noted. The test has good sensitivity (83.3%) and high specificity (95.2%) but some dogs retained positive titres after elimination of infection. The sensitivity is higher than that of direct microscopic hair examination and similar to that of fungal culture with DTM (dermatophyte test medium).     

A recombinant 31.5 kDa keratinase and a crude exo-antigen from Microsporum canis fail to protect against a homologous experimental infection in guinea pigs

A Microsporum canis recombinant 31.5 kDa keratinase and a M. canis crude exo-antigen were tested as vaccines in an experimental infection model in guinea pigs. Animals were vaccinated subcutaneously three times at two-week intervals with either the keratinase, the exo-antigen or the adjuvant alone. Cutaneous challenge was performed blindly. Both humoral and cellular-specific immune responses to M. canis antigens were evaluated every 14 days, while a blind evaluation of clinical lesion development and fungal persistency in skin were monitored weekly. Vaccination induced very high and significant (P < 0.01) antibody responses towards both antigens. High cell-mediated immune responses to both immunogens were also induced by vaccination. After challenge, however, scores reflecting the severity of dermatophytic lesions did not differ significantly between vaccinated and control groups at any time after challenge. These results suggest that, in the guinea pig, the induction of specific immune responses against the M. canis-secreted antigens used in this study are not protective against challenge exposure.     

Dysgonic strain of Microsporum canis pseudomycetoma in a Domestic Long-hair cat

A 4-year-old Domestic Long-hair cat was presented with two large non-painful, ulcerated and suppurative lesions over the flanks. Histopathology and cytology were consistent with fungal pyogranulomatous inflammation. Culture of tissue yielded a dysgonic strain of Microsporum canis. The cat was treated successfully by staged en bloc resections of the lesions, followed by oral ketoconazole, then oral terbinafine. This is the first reported case of dermatophytic pseudomycetoma in a Domestic Long-hair cat in Australia.     

Attempted treatment of tigers (Panthera tigris) infected with Microsporum canis.

An outbreak of dermatophytosis caused by Microsporum canis occurred in tigers (Panthera tigris) at an exotic felid sanctuary in 2003. In an attempt to find an effective, practical, safe, and affordable method for controlling this epizootic, a clinical treatment trial was conducted. Nonalopecic tigers were studied to address the inapparent carrier state observed at the facility. The efficacy of three topical and environmental treatment combinations of a 2% lime sulfur solution and a peroxide-based cleaner were evaluated in nonalopecic, culture-positive tigers (n = 18) housed in four separate enclosures. Lime sulfur solution was applied topically to all of these animals. As a control, nonalopecic but culture-positive tigers (n = 6) housed in two other enclosures were not treated. Environmental treatments included lime sulfur solution (n = 1), a peroxide-based cleaner (n = 1), and no treatment (n = 2). All solutions were applied at 2-wk intervals for seven treatments. The 2% lime sulfur solution treatments were unsuccessful in resolving infections in most tigers. Lime sulfur was effective in suppressing environmental fungal growth immediately posttreatment, whereas the peroxide-based cleaner was not effective. A follow-up survey of all study tigers and their enclosures was conducted 2 yr later, at which time 22 of 24 tigers (92%) had attained resolution, defined as two sequential negative hair cultures. Review of the culture results during the clinical trial and follow-up study suggests that nonalopecic dermatophytosis in tigers that are housed outdoors may not warrant aggressive individual or environmental treatment, as the infection may clear with time.     

Synergistic inhibition of the growth in vitro of Microsporum canis by miconazole and chlorhexidine

An agar dilution technique was used to assess the minimum inhibitory concentrations (MIC) of miconazole, chlorhexidine and a 1:1 combination of both agents for 10 isolates of Microsporum canis. For nine of 10 of the isolates, a combination of miconazole and chlorhexidine was more effective than either agent alone; fractional inhibitory concentration indices indicated a synergistic effect for five isolates and an additive effect for four. These results illustrate the potent antimycotic effect of miconazole and chlorhexidine against M. canis and are in accordance with previous clinical studies that showed the value of using miconazole and chlorhexidine shampoo in association with oral griseofulvin in the treatment of feline dermatophytosis caused by M. canis.     

Chitin synthase 1 (Chs1) gene sequences of Microsporum equinum and Trichophyton equinum

Chitin synthase 1 (Chs1) genes from Microsporum equinum and Trichophyton equinum were compared with those of the other dermatophytes. The Chs1 nucleotide sequences of these dermatophytes from horses showed more than 80% similarity to those of Arthroderma benhamiae, A. fulvum, A. grubyi, A. gypseum, A. incruvatum, A. otae, A. simii, A. vanbreuseghemii, Epidermophyton floccosum, T. mentagrophytes var. interdigitale (T. interdigitale), T. rubrum and T. violaceum. Especially high degree of nucleotide sequence similarity of more than 99% was noted between the Chs1 gene fragments of M. equinum and A. otae, and those of T. equinum, T. interdigitale and A. vanbreuseghemii, respectively. The phylogenetic analysis of their sequences revealed that M. equinum was genetically very close to A. otae and T. equinum to A. vanbreuseghemii. A molecular analysis of Chs1 genes will provide useful information for the genetic relatedness of M. equinum and T. equinum and confirm the value of DNA sequencing in identification of these two dermatophytes.     

Dermatophytosis and papular eosinophilic/mastocytic dermatitis (urticaria pigmentosa-like dermatitis) in three Devon Rex cats

PRESENTING SIGNS: Three Devon Rex cats were presented with multiple erythematous papules, occasionally associated with crusting and hyperpigmentation, with a linear distribution on the head, neck, chest and abdomen. One cat also had multifocal alopecia with hyperpigmentation on the dorsum. DIAGNOSIS AND TREATMENT: Clinical and histopathological features were suggestive of papular eosinophilic/mastocytic dermatitis (urticaria pigmentosa-like dermatitis). In all cases, dermatophytosis was diagnosed: in cases 1 and 2 there was histopathological evidence of dermatophytosis, while fungal culture was positive for Microsporum canis in cases 2 and 3. In all cats, lesions disappeared following antifungal treatment. CLINICAL SIGNIFICANCE: Papular eosinophilic/mastocytic dermatitis in Devon Rex cats may represent either an atypical presentation of dermatophytosis or a clinical and histological reaction pattern to various diseases, including dermatophytosis and allergic diseases. Clinical differentiation is crucial as there are important implications regarding treatment and, in particular, the use of glucocorticoids, which are contraindicated in cases of dermatophytosis.     

Development of an in vitro, isolated, infected spore testing model for disinfectant testing of Microsporum canis isolates

The isolated infected hair model is a commonly used technique to test the fungicidal efficacy of topical therapies against Microsporum canis. The most commonly used model uses mats of infective hairs, and results from various laboratories have differed. The objectives of this study were to develop a method to produce spores for testing when only mycelial forms were available and to develop a semiquantitative testing method that used only infective spores from hairs, and not pooled hair samples for testing. Ten isolates of M. canis were used in this study. Juvenile guinea pigs were easily infected using mycelial forms of M. canis and large numbers of spores were easily harvested for testing. Eight dilutions of disinfectants were tested. Fungal culture data were evaluated using an endpoint dilution at which there was 100% fungicidal activity, i.e. no growth on the plates. The 10 samples showed identical results. Chlorhexidine and Virkon(R) S were ineffective even when used at x4 the manufacturer's recommended dilution. Lime sulphur (1 : 33), enilconazole (20 microL mL(-1)), and bleach (1 : 10) were consistently effective when used at the recommended dilution. In addition, lime sulphur and enilconazole were 100% fungicidal even when the recommended concentration was diluted 1 : 4 or x4 as dilute as recommended.     

Incidence, immunity and treatment of feline dermatophytosis

Feline dermatophytosis is a superficial skin infection characterized by the invasion of cornified tissues such as hair and nails.This infection is nearly always caused by Microsporum canis. Infected animals release infective spores in the environment which will then contaminate other animals or humans. Infected animals usually develop immunity so the infection will spontaneously disappear after a few weeks to months. Long haired and immunocom-promised cats do not have the same ability to acquire resistance and spontaneous recovery does usually not occur. The treatment of such an infection will require topical and systemic treatment of all contaminated and in-contact cats. The use of desinfectants such as bleach or enilconazole has been proven effective to destroy the spores in the environment. In addition, the efficacy of topical and systemic treatments with azole derivates or allylamines has also been demonstrated in several studies. On the contrary, dermatophyte vaccination has never been proven effective in well controlled studies. Regular follow-up and fungal cultures are mandatory to ensure succesfull treatment.