Production of Fructooligosaccharides by Aspergillus phoenicis Biofilm on Polyethylene as Inert Support

Aspergillus phoenicis biofilms on polyethylene as inert support were used to produce fructooligosaccharides (FOS) in media containing 25% (m/V) of sucrose as a carbon source. The maximum production of total FOS (122 mg/mL), with 68% of 1-kestose and 32% of nystose, was obtained in Khanna medium maintained at 30 degrees C for 48 h under orbital agitation (100 rpm). At high concentrations of sucrose (30%, m/V), the recovery of FOS was higher than that observed at a low concentration (5%, m/V). High levels of FOS (242 mg/mL) were also recovered when using the biofilm in sodium acetate buffer with high sucrose concentration (50%, m/V) for 10 h. When the dried biofilm was reused in a fresh culture medium, there was a recovery of approx. 13.7% of total FOS after 72 h of cultivation at 30 C, and 10% corresponded to 1-kestose. The biofilm morphology, analyzed by scanning electron microscope, revealed a noncompact mycelium structure, with unfilled spaces and channels present among the hyphae. The results obtained in this study show that A. phoenicis biofilms may find application for FOS production in a single-step fermentation process, which is cost-effective in terms of reusability, downstream processing and efficiency.

PCR-Internal Transcribed Spacer (ITS) genes sequencing and phylogenetic analysis of clinical and environmental Aspergillus species associated with HIV-TB co infected patients in a hospital in Abeokuta, southwestern Nigeria.

Background: Aspergillosis has been identified as one of the hospital acquired infections but the contribution of water and inhouse air as possible sources of Aspergillus infection in immunocompromised individuals like HIV-TB patients have not been studied in any hospital setting in Nigeria. Objective: To identify and investigate genetic relationship between clinical and environmental Aspergillus species associated with HIV-TB co infected patients. Methods: DNA extraction, purification, amplification and sequencing of Internal Transcribed Spacer (ITS) genes were performed using standard protocols. Similarity search using BLAST on NCBI was used for species identification and MEGA 5.0 was used for phylogenetic analysis. Results: Analyses of sequenced ITS genes of selected fourteen (14) Aspergillus isolates identified in the GenBank database revealed Aspergillus niger (28.57%), Aspergillus tubingensis (7.14%), Aspergillus flavus (7.14%) and Aspergillus fumigatus (57.14%). Aspergillus in sputum of HIV patients were Aspergillus niger, A. fumigatus, A. tubingensis and A. flavus. Also, A. niger and A. fumigatus were identified from water and open-air. Phylogenetic analysis of sequences yielded genetic relatedness between clinical and environmental isolates. Conclusion: Water and air in health care settings in Nigeria are important sources of Aspergillus sp. for HIV-TB patients.

Obstrução nasal por granuloma fúngico em eqüino: relato de caso

Um eqüino de nove anos de idade apresentou ausência de ar expirado e secreção serossanguinolenta na narina direita, associado a ruído respiratório. Os exames endoscópico e radiológico mostraram uma formação de aproximadamente seis centímetros de diâmetro recoberta por mucosa amarelada, que obstruía a cavidade nasal direita e insinuava-se para a cavidade nasal esquerda. Tal massa foi ressecada por meio de sinusotomia frontal direita. O exame histológico e a cultura revelaram lesão granulomatosa causada por fungos. O tratamento pós-operatório compreendeu associação de antibiótico e antiinflamatório, assim como de lavagens com água destilada e chá de camomila.

Indoor and outdoor atmospheric fungal spores in the São Paulo metropolitan area (Brazil): species and numeric concentrations

The aim of this study was to estimate the indoor and outdoor concentrations of fungal spores in the Metropolitan Area of Sao Paulo (MASP), collected at different sites in winter/spring and summer seasons. The techniques adopted included cultivation (samples collected with impactors) and microscopic enumeration (samples collected with impingers). The overall results showed total concentrations of fungal spores as high as 36,000 per cubic meter, with a large proportion of non culturable spores (around 91 per cent of the total). Penicillium sp. and Aspergillus sp. were the dominant species both indoors and outdoors, in all seasons tested, occurring in more than 30 per cent of homes at very high concentrations of culturable airborne fungi [colony forming units(CFU) m−3]. There was no significant difference between indoor and outdoor concentrations. The total fungal spore concentration found in winter was 19 per cent higher than that in summer. Heat and humidity were the main factors affecting fungal growth; however, a non-linear response to these factors was found. Thus, temperatures below 16°C and above 25°C caused a reduction in the concentration (CFU m−3) of airborne fungi, which fits with MASP climatalogy. The same pattern was observed for humidity, although not as clearly as with temperature given the usual high relative humidity (above 70 per cent) in the study area. These results are relevant for public health interventions that aim to reduce respiratory morbidity among susceptible populations

Epidemiologic aspects and clinical outcome of fungal keratitis in southeastern Brazil

PURPOSE. Fungal keratitis (FK) is a sight-threatening disease, more prevalent in developing regions. The present retrospective study was conducted in order to evaluate the epidemiologic and clinical aspects and the progression of FK in patients treated at two ophthalmologic reference centers in Southeast Brazil. METHODS. The charts of patients with infectious keratitis treated between 2000 and 2004 were reviewed. For the 66 cases of FK confirmed by microbiological analysis, data related to patient, disease, and therapeutic approaches were obtained. RESULTS. Mean patient age was 40.7 +/- 16 years. Fifty-three were men and 13 were women. Ocular trauma occurred in 40% of cases (27). Previous medications taken by the patients were quinolone in 72.5% and antimycotics in 30%. Visual acuity (VA) at presentation was >0.3 in 16% and <0.1 in 74.5%. Penetrant keratoplasty was performed in 38% and evisceration in 15%. The causing agents were Fusarium sp in 67%, Aspergillus sp in 10.5%, and Candida sp in 10%. Medication alone resolved 39% of cases within a mean period of 24.5 +/- 12 days. Final VA was >0.3 in 28%, and <0.1 in 63%. CONCLUSIONS. Fungal keratitis presented as a disease with severe complications, predominantly among young males, and was mostly caused by filamentous fungi. The present information permits the establishment of preventive strategies. Reducing the time between onset and treatment and using more accessible specific medication would reverse the negative prognosis. (Eur J Ophthalmol 2009; 19: 355-61)

Prevalence of fungi in indoor air with reference to gymnasiums with swimming pools

Fungal contamination of air in 10 gymnasiums with swimming pools was monitored. Fifty air samples of 200 L each were collected, using a Millipore air tester, from the area surrounding the pool, in training studios, in showers and changing rooms for both sexes, and also, outside premises, since these are the places regarded as reference. Simultaneously, environmental parameters – temperature and humidity – were also monitored. Some 25 different species of fungi were identified. The six most commonly isolated genera were the following: Cladosporium sp. (36.6%), Penicillium sp. (19.0%), Aspergillus sp. (10.2%), Mucor sp. (7%), Phoma sp. and Chrysonilia sp. (3.3%). For yeasts, three different genera were identified, namely, Rhodotorula sp. (70%), Trichosporon mucoides and Cryptococcus uniguttulattus (10%).

Fungal contamination of poultry litter: a public health problem

Although numerous studies have been conducted on microbial contaminants associated with various stages related to poultry and meat products processing, only a few reported on fungal contamination of poultry litter. The goals of this study were to (1) characterize litter fungal contamination and (2) report the incidence of keratinophilic and toxigenic fungi presence. Seven fresh and 14 aged litter samples were collected from 7 poultry farms. In addition, 27 air samples of 25 litters were also collected through impaction method, and after laboratory processing and incubation of collected samples, quantitative colony-forming units (CFU/m3) and qualitative results were obtained. Twelve different fungal species were detected in fresh litter and Penicillium was the most frequent genus found (59.9%), followed by Alternaria (17.8%), Cladosporium (7.1%), and Aspergillus (5.7%). With respect to aged litter, 19 different fungal species were detected, with Penicillium sp. the most frequently isolated (42.3%), followed by Scopulariopsis sp. (38.3%), Trichosporon sp. (8.8%), and Aspergillus sp. (5.5%). A significant positive correlation was found between litter fungal contamination (CFU/g) and air fungal contamination (CFU/m3). Litter fungal quantification and species identification have important implications in the evaluation of potential adverse health risks to exposed workers and animals. Spreading of poultry litter in agricultural fields is a potential public health concern, since keratinophilic (Scopulariopsis and Fusarium genus) as well as toxigenic fungi (Aspergillus, Fusarium, and Penicillium genus) were isolated.

Fungal contamination in two Portuguese wastewater treatment plants

The presence of filamentous fungi was detected in wastewater and air collected at wastewater treatment plants (WWTP) from several European countries. The aim of the present study was to assess fungal contamination in two WWTP operating in Lisbon. In addition, particulate matter (PM) contamination data was analyzed. To apply conventional methods, air samples from the two plants were collected through impaction using an air sampler with a velocity air rate of 140 L/min. Surfaces samples were collected by swabbing the surfaces of the same indoor sites. All collected samples were incubated at 27°C for 5 to 7 d. After lab processing and incubation of collected samples, quantitative and qualitative results were obtained with identification of the isolated fungal species. For molecular methods, air samples of 250 L were also collected using the impinger method at 300 L/min airflow rate. Samples were collected into 10 ml sterile phosphate-buffered saline with 0.05% Triton X-100, and the collection liquid was subsequently used for DNA extraction. Molecular identification of Aspergillus fumigatus and Stachybotrys chartarum was achieved by real-time polymerase chain reaction (RT-PCR) using the Rotor-Gene 6000 qPCR Detection System (Corbett). Assessment of PM was also conducted with portable direct-reading equipment (Lighthouse, model 3016 IAQ). Particles concentration measurement was performed at five different sizes: PM0.5, PM1, PM2.5, PM5, and PM10. Sixteen different fungal species were detected in indoor air in a total of 5400 isolates in both plants. Penicillium sp. was the most frequently isolated fungal genus (58.9%), followed by Aspergillus sp. (21.2%) and Acremonium sp. (8.2%), in the total underground area. In a partially underground plant, Penicillium sp. (39.5%) was also the most frequently isolated, also followed by Aspergillus sp. (38.7%) and Acremonium sp. (9.7%). Using RT-PCR, only A. fumigatus was detected in air samples collected, and only from partial underground plant. Stachybotrys chartarum was not detected in any of the samples analyzed. The distribution of particle sizes showed the same tendency in both plants; however, the partially underground plant presented higher levels of contamination, except for PM2.5. Fungal contamination assessment is crucial to evaluating the potential health risks to exposed workers in these settings. In order to achieve an evaluation of potential health risks to exposed workers, it is essential to combine conventional and molecular methods for fungal detection. Protective measures to minimize worker exposure to fungi need to be adopted since wastewater is the predominant internal fungal source in this setting.

Fungal contamination assessment in Portuguese elderly care centers

Individuals spend 80-90% of their day indoors and elderly subjects are likely to spend even a greater amount of time indoors. Thus, indoor air pollutants such as bioaerosols may exert a significant impact on this age group. The aim of this study was to characterize fungal contamination within Portuguese elderly care centers. Fungi were measured using conventional as well as molecular methods in bedrooms, living rooms, canteens, storage areas, and outdoors. Bioaerosols were evaluated before and after the microenvironments' occupancy in order to understand the role played by occupancy in fungal contamination. Fungal load results varied from 32 colony-forming units CFU m(-3) in bedrooms to 228 CFU m(-3) in storage areas. Penicillium sp. was the most frequently isolated (38.1%), followed by Aspergillus sp. (16.3%) and Chrysonilia sp. (4.2%). With respect to Aspergillus genus, three different fungal species in indoor air were detected, with A. candidus (62.5%) the most prevalent. On surfaces, 40 different fungal species were isolated and the most frequent was Penicillium sp. (22.2%), followed by Aspergillus sp. (17.3%). Real-time polymerase chain reaction did not detect the presence of A. fumigatus complex. Species from Penicillium and Aspergillus genera were the most abundant in air and surfaces. The species A. fumigatus was present in 12.5% of all indoor microenvironments assessed. The living room was the indoor microenvironment with lowest fungal concentration and the storage area was highest.

Avaliação da exposição a fungos e partículas em explorações avícolas e suinícolas: estudo

O projeto “Avaliação da Exposição a Fungos e Partículas em Explorações Avícolas e Suinícolas” contemplou um elevado número de colheitas ambientais e biológicas e respectivo processamento laboratorial, sendo apenas possível a sua concretização graças ao financiamento disponibilizado pela Autoridade para as Condições de Trabalho. Foi realizado um estudo transversal para avaliar a contaminação causada por fungos e partículas em 7 explorações avícolas e 7 explorações suinícolas. No que concerne à monitorização biológica, foram medidos os parâmetros espirométricos, utilizando o espirómetro MK8 Microlab, avaliada a existência de sintomas clínicos associados com a asma e outras doenças alérgicas, através de questionário adaptado European Community Respiratory Health Survey e, ainda, avaliada a sensibilização aos agentes fúngicos (IgE). Foram ainda adicionados dois objetivos ao estudo, designadamente: aferir a existência de três espécies/estirpes potencialmente patogénicas/toxinogénicas com recurso à biologia molecular e avaliar a exposição dos trabalhadores à micotoxina aflatoxina B1 por recurso a indicador biológico de exposição. Foram colhidas 27 amostras de ar de 25 litros nas explorações avícolas e 56 de 50 litros nas explorações suinícolas através do método de impacto. As colheitas de ar e a medição da concentração das partículas foram realizadas no interior e no exterior dos pavilhões, sendo este último considerado como local de referência. Simultaneamente, a temperatura e a humidade relativa também foram registadas. As colheitas das superfícies foram realizadas através da técnica de zaragatoa, tendo sido utilizado um quadrado de metal inoxidável de 10 cm de lado, de acordo com a International Standard ISO 18593 – 2004. As zaragatoas obtidas (20 das explorações avícolas e 48 das explorações suinícolas) foram inoculadas em malte de extract agar (2%) com cloranfenicol (0,05 g/L). Além das colheitas de ar e de superfícies, foram também obtidas colheitas da cama das explorações avícolas (7 novas e 14 usadas) e da cobertura do pavimento das explorações suinícolas (3 novas e 4 usadas) e embaladas em sacos esterilizados. Cada amostra foi diluída e inoculada em placas contendo malte extract agar. Todas as amostras foram incubadas a 27,5ºC durante 5 a 7 dias e obtidos resultados quantitativos (UFC/m3; UFC/m2; UFC/g) e qualitativos com a identificação das espécies fúngicas. Para a aplicação dos métodos de biologia molecular foram realizadas colheitas de ar de 300 litros utilizando o método de impinger com a velocidade de recolha de 300 L/min. A identificação molecular de três espécies potencialmente patogénicas e/ou toxinogénicas (Aspergillus flavus, Aspergillus fumigatus e Stachybotrys chartarum) foram obtidas por PCR em tempo real (PCR TR) utilizando o Rotor-Gene 6000 qPCR Detection System. As medições de partículas foram realizadas por recurso a equipamento de leitura direta (modelo Lighthouse, 2016 IAQ). Este recurso permitiu medir a concentração (mg/m3) de partículas em 5 dimensões distintas (PM 0.5; PM 1.0; PM 2.5; PM 5.0; PM10). Nas explorações avícolas, 28 espécies/géneros de fungos foram isolados no ar, tendo Aspergillus versicolor sido a espécie mais frequente (20.9%), seguida por Scopulariopsis brevicaulis (17.0%) e Penicillium sp. (14.1%). Entre o género Aspergillus, Aspergillus flavus apresentou o maior número de esporos (>2000 UFC/m3). Em relação às superfícies, A. versicolor foi detetada em maior número (>3 × 10−2 UFC/m2). Na cama nova, Penicillium foi o género mais frequente (59,9%), seguido por Alternaria (17,8%), Cladosporium (7,1%) e Aspergillus (5,7%). Na cama usada, Penicillium sp. foi o mais frequente (42,3%), seguido por Scopulariopsis sp. (38,3%), Trichosporon sp. (8,8%) e Aspergillus sp. (5,5%). Em relação à contaminação por partículas, as partículas com maior dimensão foram detectadas em maiores concentrações, designadamente as PM5.0 (partículas com a dimensão de 5.0 bm ou menos) e PM10 (partículas com a dimensão de 10 bm ou menos). Neste setting a prevalência da alteração ventilatória obstrutiva foi superior nos indivíduos com maior tempo de exposição (31,7%) independentemente de serem fumadores (17,1%) ou não fumadores (14,6%). Relativamente à avaliação do IgE específico, foi apenas realizado em trabalhadores das explorações avícolas (14 mulheres e 33 homens), não tendo sido encontrada associação positiva (p<0.05%) entre a contaminação fúngica e a sensibilização a antigénios fúngicos. No caso das explorações suinícolas, Aspergillus versicolor foi a espécie mais frequente (20,9%), seguida por Scopulariopsis brevicaulis (17,0%) e Penicillium sp. (14,1%). No género Aspergillus, A. versicolor apresentou o maior isolamento no ar (>2000 UFC/m3) e a maior prevalência (41,9%), seguida por A. flavus e A. fumigatus (8,1%). Em relação às superfícies analisadas, A. versicolor foi detetada em maior número (>3 ×10−2 UFC/m2). No caso da cobertura do pavimento das explorações suinícolas, o género Thicoderma foi o mais frequente na cobertura nova (28,0%) seguida por A. versicolor e Acremonium sp. (14,0%). O género Mucor foi o mais frequente na cobertura usada (25,1%), seguido por Trichoderma sp. (18,3%) e Acremonium sp. (11,2%). Relativamente às partículas, foram evidenciados também valores mais elevados na dimensão PM5 e, predominantes nas PM10. Neste contexto, apenas 4 participantes (22,2%) apresentaram uma alteração ventilatória obstrutiva. Destes, as obstruções mais graves encontraram-se nos que também apresentavam maior tempo de exposição. A prevalência de asma na amostra de trabalhadores em estudo, pertencentes aos 2 contextos em estudo, foi de 8,75%, tendo-se verificado também uma prevalência elevada de sintomatologia respiratória em profissionais não asmáticos. Em relação à utilização complementar dos métodos convencionais e moleculares, é recomendável que a avaliação da contaminação fúngica nestes settings, e, consequentemente, a exposição profissional a fungos, seja suportada pelas duas metodologias e, ainda, que ocorre exposição ocupacional à micotoxina aflatoxina B1 em ambos os contextos profissionais. Face aos resultados obtidos, é importante salientar que os settings alvo de estudo carecem de uma intervenção integrada em Saúde Ocupacional no âmbito da vigilância ambiental e da vigilância da saúde, com o objetivo de diminuir a exposição aos dois factores de risco estudados (fungos e partículas).

Isolamento de fungos filamentosos em água utilizada em uma unidade de hemodiálise

A despeito da relativa freqüência de infecções fúngicas oportunísticas em pacientes sob hemodiálise, os reservatórios ambientais destes permanecem desconhecidos, embora alguns estudos recentes tenham correlacionado o suprimento de água como fonte desses microrganismos. O objetivo deste trabalho foi monitorar a qualidade micológica do sistema hídrico de uma Unidade de Hemodiálise, do interior do Estado de São Paulo, Brasil, no período entre abril e julho de 2006. Foram coletadas amostras (15), de 1000mL em 7 pontos de distribuição de água empregando-se técnica da membrana filtrante (0,45µm). Foram isolados 116 fungos filamentosos, dos quais 47 (40,5%) Trichoderma sp, 29 (25%) Cladosporium sp, 16 (13,8%) Aspergillus sp e 11 (9,5%) Fusarium sp. Mediante os resultados, sugerimos que suprimentos de água para Unidades de Hemodiálise devam ser monitorados também quanto ao aspecto micológico, adotando-se medidas profiláticas eficazes que minimizem a exposição destes pacientes imunodeficientes a fontes aquáticas ambientais contaminadas.

Eco-epidemiologic study of emerging fungi related to the work of babaçu coconut breakers in the State of Maranhão, Brazil

Introduction: There are more than 300,000 extractors using the babaçu coconut as a source of income in the States of Maranhão, Pará, Tocantins and Piauí, and this activity is associated with fungal infections. The objective of this study was to examine the occurrence of emergent fungi in the conjunctiva, nails and surface and subcutaneous injuries of female coconut breakers in Esperantinópolis, Maranhão. Additionally, soil samples and palm structures were collected. Methods: The obtained samples were cultured in Petri dishes containing potato-dextrose-agar and chloramphenicol. The etiological agent was confirmed by a direct mycological exam and growth in culture. Results: In total, 150 domiciles were visited, and samples were collected from 80 patients. From the ground, the most frequently isolated fungus was Aspergillus niger (53. 8%). the most frequently detected fungus in babaçu coconut was Aspergillus niger (66.7%). Conjunctival fungal growth occurred in 76.3% of the women. The ocular fungal microbiota consisted of filamentous fungi (80.6%), and yeasts were present in 19.4% of cases. Onychomycosis was diagnosed in 44% (11/25) of the women. Conclusions: The identification of the genera Neosartorya, Rhizopus and Curvularia in onychomycoses shows that emergent filamentous fungi can be isolated. Aspergillus sp., Penicillium sp. and Scedosporium sp. were the predominant genera found in the babaçu coconut. From ocular conjunctiva, Candida spp. were the most prevalent species isolated, and Fusarium sp. was present only in one woman. The nearly permanent exposure of coconut breakers to the external environment and to the soil is most likely the reason for the existence of a mycotic flora and fungal infections, varying according to the individual's practices and occupation.

Seleção de fungos produtores de β-D-frutosiltransferase por fermentação em estado sólido

A enzima β-D-frutosiltransferase é responsável pela síntese de FOS (frutooligossacarídeos) a partir de sacarose por reação de transfrutosilação é produzida por diferentes micro-organismos, principalmente por fungos filamentosos. O objetivo deste trabalho foi selecionar a melhor linhagem fúngica produtora da -D-frutosiltransferase por fermentação em estado sólido, bem como o método de extração. A fermentação em estado sólido utilizando o substrato farelo de trigo umedecido com solução de sacarose atingindo 70% de umidade na concentração de esporos de 107 no tempo de 96 horas de crescimento. Todas as linhagens manipuladas apresentaram atividade hidrolítica, no entanto apenas uma linhagem não demonstrou atividade transfrutosilação. O isolado SIS 14 que pertence ao gênero Aspergillus sp. destacou-se pelos maiores valores em atividade no método de extração utilizando água destilada, apresentando 300,90 U/mL na atividade de transfrutosilação e na atividade hidrolítica de 155,74 U/mL. Contudo, pode-se perceber que dos solventes estudados a água destilada foi melhor obtendo o valor em atividade de transfrutosilação, como também a linhagem SIS 14 é promissora para a produção da β-D-frutosiltransferase.

Superação de dormência na qualidade de sementes e mudas: influência na produção de Senna multijuga (L. C. Rich.) Irwin & Barneby

O objetivo deste trabalho foi determinar a qualidade fisiológica e sanitária de sementes de Senna multijuga(L. C. Rich.) Irwin & Barneby relacionada aos métodos de superação de dormência e à interferência na produção de mudas. As sementes foram submetidas aos seguintes métodos: imersão em água fervente, as sementes foram imersas em água, com temperatura de 100°C, até esfriar, por 24 horas; escarificação ácida, onde as sementes foram imersas em ácido sulfúrico (H2SO4) a 90%, por 10 e 20 minutos, e testemunha (sem tratamento). Foram realizados os testes de sanidade, germinação, tetrazólio e avaliação da qualidade das mudas. O delineamento experimental foi inteiramente casualizado. Para a avaliação da germinação foi utilizado um esquema fatorial (4 X 2), com quatro métodos de superação de dormência X dois fotoperíodos, para os substratos rolo-de-papel e vermiculita. A escarificação ácida constituiu-se no método mais eficiente para a superação da dormência das sementes de Senna multijuga. Penicillium sp. e Aspergillus sp. tiveram sua incidência aumentada quando o tegumento foi danificado pela escarificação ácida por 20 minutos. O controle de Fusarium spp. aumentou gradativamente com o aumento do tempo de exposição ao ácido sulfúrico.

Onychomycosis insensitive to systemic terbinafine and azole treatments reveals non-dermatophyte moulds as infectious agents.

BACKGROUND: Dermatophytes are the main cause of onychomycoses, but various non-dermatophyte filamentous fungi are often isolated from abnormal nails. OBJECTIVE: Our aim was the in situ identification of the fungal infectious agent in 8 cases of onychomycoses which could not be cured after systemic terbinafine and itraconazole treatment. METHODS: Fungal DNA was extracted from nail samples, and infectious fungi were identified by restriction fragment length polymorphism (RFLP) of amplified fungal ribosomal DNA using a previously described PCR/RFLP assay. RESULTS: PCR/RFLP identification of fungi in nails allows the identification of the infectious agent: Fusarium sp., Acremonium sp. and Aspergillus sp. were found as a sole infectious agent in 5, 2 and 1 cases, respectively. CONCLUSIONS: Fusarium spp. and other non-dermatophyte filamentous fungi are especially difficult to cure in onychomycoses utilising standard treatment with terbinafine and itraconazole. PCR fungal identification helps demonstrate the presence of moulds in order to prescribe alternative antifungal treatments.