Histoplasmosis in Europe: report on an epidemiological survey from the European Confederation of Medical Mycology Working Group.

The purpose of this survey was to systematically collect data on individuals with histoplasmosis in Europe over a 5-year period (from January 1995 to December 1999). This included information on where and how the infection was acquired, the patient's risk factors, the causative organism, how the infection was diagnosed and what therapy the patients received. Data were sent on a standardized survey form via a national convenor to the coordinator. During the survey, 118 cases were reported, with 62 patients having disseminated disease, 31 acute pulmonary infection, chronic pulmonary infection in 6 and localized disease in 2 patients. For 17 patients, the diagnosis of histoplasmosis was incidental, usually secondary to investigations for lung cancer. Most patients had travelled to known endemic areas, but 8 patients (from Italy, Germany and Turkey) indicated that they had not been outside their countries of origin and hence these cases appear to be autochthonous. Notable observations during the survey were the reactivation of the disease up to 50 years after the initial infection in some patients and transmission of the infection by a transplanted liver. Itraconazole was the most commonly used therapy in both pulmonary and disseminated disease. The observation of autochthonous cases of disease suggests that the endemic area of histoplasmosis is wider than classically reported and supports continued surveillance of the disease throughout Europe.

Testing of antifungal combinations against yeasts and dermatophytes.

BACKGROUND: Fungal infections of the nail are a common and chronic problem. The main pathogens responsible for onychomycosis are dermatophytes, yeasts and moulds. Despite significant improvements, approximately 20% of patients with onychomycosis still fail on antifungal therapy. The successful exploitation of drug synergy may provide a useful approach to improve cure rates. METHODS: The minimum inhibitory concentrations (MIC(80)) were recorded for pathogens that are most frequently responsible for onychomycosis against combinations of several antifungal agents, namely, fluconazole, itraconazole, terbinafine and amorolfine. Fractional inhibitory concentrations (FICs) were then calculated from the MIC(80) results and the FIC values for each drug in the combinations added to determine the degree of synergy. A combined value of <1 was taken to suggest synergy; a value of 1-2 indicated an additive effect or indifference; and a combined FIC value of >2 was taken to suggest antagonism. RESULTS: Overall, 46% of amorolfine combinations showed results suggestive of synergy, with the most synergistic results seen against dermatophytes (54%) and moulds (52%). CONCLUSIONS: Some combinations of drugs may have synergistic activity in vitro; however, the importance of this in a clinical setting is yet to be established, and more studies are justified.

Drug synergies and the potential for combination therapy in onychomycosis.

Synergy between antifungal drugs is well recognized. However, combinations of antifungals are yet to be widely adopted to treat onychomycosis. The rationale for the combination of topical and oral therapy in the treatment of onychomycosis is that the systemic antifungal reaches the infection area via the nail bed and the topical agent is absorbed through the nail surface. The combination of antifungals used should ideally be synergistic in their mode of action. Synergy between amorolfine and other antifungals has been demonstrated in vitro. This is presumably due to differences in the precise point of action of the drugs on the fungal cell membrane, where they inhibit the synthesis of ergosterol. Thus, combinations can result in increased antifungal activity at lower concentrations of both drugs. The potential exists therefore for combinations of antifungals to achieve higher cure rates in onychomycosis, in a shorter time than is currently possible. This approach warrants further investigation.

Prediction of outcome in the treatment of onychomycosis.

Patients with toenail onychomycosis remain a therapeutic challenge despite the introduction of new systemic therapies. Around 20% of patients remain uncured even with optimal oral therapy, but the reasons for treatment failure are unclear. Thus far there are no data to suggest that treatment failures can be identified on the basis of their presenting features or progress during treatment. In a series of patients, we have attempted to identify clinical parameters that determine the patient response to 12 weeks of oral terbinafine for confirmed dermatophyte onychomycosis. As part of a dose-defining randomized multicentre study, 35 patients were followed for 48 weeks. The unaffected nail length, growth rate, hyperkeratosis, onycholysis and presence of a dermatophytoma were assessed prospectively. To confirm our findings, at the end of the study period we analysed retrospectively photographs that had been taken regularly throughout the trial. The average degree of hyperkeratosis was less severe in the group achieving a disease-free nail, meaning clinical and mycological cure. For mycological cure alone, no predictive factors could be identified.

Molecular strain typing of Trichophyton rubrum indicates multiple strain involvement in onychomycosis.

BACKGROUND: Trichophyton rubrum is an important cause of onychomycosis. Molecular strain typing methods have recently been developed to address questions of epidemiology and source of relapse following treatment. OBJECTIVES: To determine whether T. rubrum nail infections are caused by one or more strains of this fungus. METHODS: Nail specimens from 10 patients with onychomycosis due to T. rubrum were cultured and five colonies per culture plate were selected for molecular strain typing. DNA was extracted from these isolates and subjected to a polymerase chain reaction-based typing method that analyses variations in numbers of repetitive elements in the non-transcribed spacer region of the ribosomal RNA gene repeats. RESULTS: In six of 10 specimens, there were two or more T. rubrum strain types present. CONCLUSIONS: This preliminary study suggests that in many cases of fungal nail infection by T. rubrum, multiple strains are involved. This has important implications for epidemiological studies and possibly for therapy.

Prognostic factors of mycological cure following treatment of onychomycosis with oral antifungal agents.

BACKGROUND: There is considerable literature on the efficacy and safety of various drugs used in treating onychomycosis; however, little information is available regarding prognostic factors which may be associated with non-response to conventional treatment. OBJECTIVES: To identify parameters influencing mycological cure at 72 weeks following treatment of toenail onychomycosis with oral antifungal agents. METHODS: Univariate and multivariate logistic regression analysis from a randomized double-blind controlled trial including 496 patients with toenail onychomycosis caused by dermatophytes. RESULTS: Baseline parameters including patient's age, gender, weight, number of toenails involved, percentage of nail involvement, duration of infection, history of previous treatment were not associated with mycological cure. In the multivariate prognostic factor analysis based on factors assessed at week 12, positive mycological culture at 12 weeks [odds ratio (OR): 0.583; 95% confidence interval (CI): 0.370-0.918] was negatively associated with mycological cure at 72 weeks. Similarly, in the multivariate prognostic factor analysis based on factors assessed at week 24, positive direct microscopy at 24 weeks (OR: 0.373; 95% CI: 0.211-0.659) and mycological culture at 24 weeks (OR: 0.293; 95% CI: 0.168-0.513) were negatively associated with mycological cure at 72 weeks. CONCLUSIONS: Mycological culture at 12 and 24 weeks and direct microscopic examination at 24 weeks can help in early identification of patients failing to respond to conventional oral antifungal treatment.

A two year global evaluation of the susceptibility of Candida species to fluconazole by disk diffusion.

The in-vitro activity of fluconazole against 46,831 yeast isolates collected over a two-year period from 57 laboratories in 33 countries worldwide was assessed using a disc diffusion method. Candida albicans was the organism isolated most frequently, accounting for 68.6% of the total number of isolates. C. glabrata, C. tropicalis, C parapsilosis and C. krusei and Cryptococcus neoformans represented 9.9, 4.7, 4.3, 1.9, and 1.4% of isolates respectively during the 2 year period and rates varied markedly between countries. In 1999 data blood isolates represented 4.9% of all isolates and intensive care unit isolates represented 9.9%. In both the 1998 and 1999 data, 99% of C. albicans were fully susceptible (S) to fluconazole, and 95.6% of all species of yeasts tested were S or susceptible-dose dependent (S-DD) to fluconazole. No emerging trends of resistance were noted with any of the Candida spp. tested as 96% of all isolates retained susceptibility (S or S-DD) to this agent.

An investigation into the pathogenesis of vulvo-vaginal candidosis.

OBJECTIVE: To monitor yeasts isolated from women during and between episodes of recurrent vulvo-vaginal candidosis (VVC) to determine whether vaginal relapse or re-infection occurred. METHODS: Women presenting at the genitourinary medicine clinic with signs and symptoms of VVC were recruited to the study (n = 121). A vaginal washing, high vaginal swab (HVS) and rectal swab were taken and the women treated with a single 500 mg clotrimazole pessary. Women were asked to re-attend after 1, 4, and 12 weeks, or when the VVC recurred, when vaginal washings and HVS were repeated. Candida isolates recovered were strain typed using the Ca3 probe and their similarity assessed. Antifungal susceptibility to fluconazole and clotrimazole were determined. RESULTS: Of the women recruited, 47 completed the study, either returning for four visits or suffering a recurrence during the study period. Of the 22 women who experienced recurrence, the same strain was responsible for the initial and recurrent episode in 17 women. For the remaining five women, four had strain replacement and one had a change of species. None of the isolates recovered from the women demonstrated resistance to either clotrimazole or fluconazole. CONCLUSIONS: Our findings support the theory of vaginal relapse and thus may support the use of more prolonged courses of antifungal therapy initially to increase the chances of eradication of the yeast.

Current and future approaches to antimycotic treatment in the era of resistant fungi and immunocompromised hosts.

Due to the ever-increasing number of immunocompromised patients, both localised and life-threatening systemic fungal infections are on the increase. Conventional treatment is of limited help, not in the least due to a less optimum benefit-to-risk ratio. Moreover, emerging pathogens with reduced antimicrobial susceptibility and the development of resistance in Candida albicans form a new challenge. Fortunately, conventional antimycotics have been improved and entirely new ones are on the horizon as well as alternative approaches such as immunoreconstitution.

The rationale for combination therapy.

Fungal infections of the nail are common in European populations. There are a number of clinical presentations usually resulting from an infection by one of the dermatophyte species, notably Trichophyton rubrum. A number of modern treatment strategies are available and are generally well tolerated and effective. However, a significant proportion of patients, 20-30%, can expect treatment failure and/or relapse following treatment. The search for new treatment modalities and drugs is hampered by our lack of understanding of the basic pathophysiological mechanisms that underlie these frequently encountered infections. A correct diagnosis is key to the implementation of successful treatment. Even then, some infections do appear to be more recalcitrant than others. This may be the result of a number of interacting factors: patient susceptibility, fungal growth patterns that resist treatment and the occurrence of dormant fungal spores (arthrospores) in the nail. Increasingly, research is turning towards the identification of clinical indicators of poor prognosis and the development of new treatment strategies to overcome them. The combination of drugs to produce synergistic activity is one possible advance towards achieving higher cure rates.

Strain identification of Trichophyton rubrum by specific amplification of subrepeat elements in the ribosomal DNA nontranscribed spacer.

Trichophyton rubrum is the commonest cause of dermatophytosis of skin and nail tissue. Molecular characterization of the T. rubrum ribosomal DNA nontranscribed-spacer region revealed two novel tandemly repetitive subelements (TRSs): TRS-1, containing a 27-bp palindromic sequence, and TRS-2. Specific amplification of TRS-1 produced strain-characteristic banding patterns (PCR types), with 21 TRS-1 PCR types recognized from 101 clinical isolates. Four simple patterns representing 1 to 4 copies of TRS-1 accounted for 75 (75%) of all 101 strains, whereas more complex patterns were observed for 21 (20%) of the 101 isolates. The copy number of TRS-2 was 0 to 3 repeats per cistron, with a majority of isolates having two copies of this element. Eleven isolates were polymorphic for TRS-2, and in combination, 23 separate PCR types were recognized by amplification of both TRS-1 and TRS-2. The PCR patterns from both elements were stable and reproducible. Elements with homology to TRS-1 were present in three phylogenetically related species, Trichophyton violaceum, Trichophyton gourvilii, and Trichophyton soudanense, but these elements were not identified in other dermatophyte taxa. There was no clear correlation of PCR type with specimen (skin or nail tissue), but certain PCR types appeared to show a bias in geographic distribution. This new method of typing T. rubrum will enable important questions about pathogenesis and epidemiology of this fungus to be addressed.

Trichophyton tonsurans tinea capitis and tinea corporis: treatment and follow-up of four affected family members.

We report a Caucasian family of two veterinary practitioners and their two children, ages 2 years and 6 months, simultaneously infected with the dermatophyte Trichophyton tonsurans, causing tinea capitis and tinea corporis in the children and tinea corporis in the parents. The parents and older child were successfully treated with oral terbinafine. The infant clinically responded to treatment with topical terbinafine and ketoconazole shampoo but presented with recurrent tinea capitis 12 months later, from which T. tonsurans was cultured. At this time, scalpbrush samples from the other family members failed to culture any fungi, and neither were fungi isolated from the family hairbrushes. The infant then received oral terbinafine, resulting in clinical and mycologic cure. After a further 12 months follow-up, there has been no mycologic evidence of recurrence in any family member.

Non-tuberculous mycobacterial lung infection complicated by chronic necrotising pulmonary aspergillosis.

We report four cases of pulmonary mycobacterial disease (three due to Mycobacterium malmoense and one to Myco- bacterium avium intracellulare) complicated by the development of chronic necrotising pulmonary aspergillosis. Difficulties with treatment and the potential benefits of steroids are discussed.

Training medical myocologists in developing countries.

Although there has over recent years been a marked rise in the incidence of serious fungal infections, many of which are prevalent in developing countries, few facilities exist for diagnosis and research in medical mycology. In most countries, medical mycology is not taught adequately to medical students and consequently there is little awareness of the importance of fungal infections. Model teaching programmes need to be developed. Practical knowledge of mycoses, their diagnosis and treatment and also basic mycology can be disseminated through well-constructed courses and workshops. Formalized training in mycology research also needs to be introduced. To achieve all of this, expertise and additional resources need to be made available. In this regard, ISHAM can and should help.

Vaginal yeasts in the era of "over the counter" antifungals.

OBJECTIVE: To establish whether there has been any rise in the prevalence of non-albicans Candida species isolated from vaginal swabs since the introduction of "over the counter" antifungal treatments. METHOD: A retrospective review looking at all positive vaginal yeast isolates collected from women attending one genitourinary medicine clinic during the 6 year period from 1993 to 1998 inclusive. All positive vaginal yeast isolates were included, regardless of whether or not the patients were symptomatic. Isolates from HIV positive women were excluded from the analysis. RESULTS: No increase in non-albicans vaginal yeast isolates was shown during the period studied. The proportion of non-albicans yeasts remained constant at approximately 5% of the total yeasts isolated. The most common non-albicans yeast isolated was C glabrata. CONCLUSION: There is no evidence from this study to suggest that the increasing use of "over the counter" antifungal treatment has selected for atypical, possibly inherently azole resistant, strains of vaginal yeasts in HIV seronegative women.

Microbiological and molecular diagnosis of deep localized cutaneous infection with Trichophyton mentagrophytes.

We describe a healthy young woman with a localized deep dermal infection on the right side of the chest wall. It was caused by the dermatophyte Trichophyton mentagrophytes, and resolved after two pulses of oral itraconazole 200 mg twice daily for 1 week. As cultural and microscopic features did not enable a precise identification of the fungus, molecular investigation was undertaken. Patterns of HaeIII restriction digests of genomic DNA from the culture matched those from Arthroderma incurvata and A. benhamiae, which is the teleomorph of T. mentagrophytes var. mentagrophytes.

Determination of optimum growth conditions for gliotoxin production by Aspergillus fumigatus and development of a novel method for gliotoxin detection.

Gliotoxin is a toxic metabolite of Aspergillus fumigatus Fresenius and other fungi. It has been suggested that this toxin may play an important role in the pathogenesis of aspergillosis as gliotoxin has immunosuppressive activity both in vitro and in vivo. We have determined the optimum growth conditions for the production of gliotoxin by selected isolates of A. fumigatus using a number of defined media. Gliotoxin was detected by thin layer chromatography and high performance liquid chromatography. The carbohydrate source, concentration of carbohydrate in the growth medium and incubation temperature were all found to influence gliotoxin production. Optimum growth conditions for gliotoxin production in our study were Czapek-Dox broth containing 30% glucose and incubation at 37 degrees C. Most of the gliotoxin was produced after 29 h incubation, during the exponential phase of growth. A novel method for screening large numbers of A. fumigatus isolates for gliotoxin production, which is both quick and easy, has also been developed, based on the ability of gliotoxin to inhibit the adherence of lung fibroblast (L929) cells to plastic microtitre plates.