Ricardo Araujo

Variability of Germinative Potential among Pathogenic Species of Aspergillus

ABSTRACT The objective of our study was to evaluate parameters influencing the germination of Aspergillus conidia. Inoculum concentration and age significantly influenced germination. Different incubation temperatures revealed significant differences among Aspergillus species. The internal human milieu provides the ideal conditions for the development of invasive disease by Aspergillus fumigatus but restricts invasion by Aspergillus flavus and Aspergillus niger.

Rapid identification of Aspergillus fumigatus within the section Fumigati.

BackgroundNew fungal species that are morphologically similar to Aspergillus fumigatus were recently described and included in section Fumigati. Misidentification of such fungal species, particularly of the human pathogens, Aspergillus lentulus, Neosartorya fischeri, Neosartorya hiratsukae, Neosartorya pseudofischeri and Neosartorya udagawae, has been increasingly reported by numerous clinical labs. Nevertheless, A. fumigatus still accounts for more than 90% of all invasive aspergillosis cases. The purpose of the present study was to develop a rapid method for the molecular identification of A. fumigatus to distinguish it from other species within the section Fumigati.ResultsA multiplex PCR was developed using prior information based on β-tubulin (βtub) and rodlet A (rodA) partial gene sequences. PCR amplification of βtub and rodA fragments resulted in a distinctive electrophoretic pattern in A. fumigatus and N. udagawae. The polymorphisms found in the smallest amplified sequence of βtub (153 bp) and rodA (103 bp) genes were then compared among and within species of this taxonomic section. βtub was able to differentiate among 13 individual species and two groups of species that included the pathogenic fungus A. lentulus. A more limited number of sequences were available for rodA; nevertheless, we were able to distinguish Aspergillus viridinutans, N. hiratsukae and N. udagawae.ConclusionsThe assay described in the present study proved to be specific and highly reproducible, representing a fast and economic way of targeting molecular identification of the relevant mould, A. fumigatus, in clinical laboratories.

Simple and highly discriminatory microsatellite-based multiplex PCR for Aspergillus fumigatus strain typing.

The answers to important questions concerning Aspergillus fumigatus pathogenicity, transmissions routes and efficacy of treatments require highly discriminating and reproducible genotyping methods. The present study was aimed at improving microsatellite methodology for A. fumigatus typing by reducing the task of strain identification to a single multiplex reaction and by selecting highly accurate short tandem repeat polymorphisms. A set of eight primer pairs was used for the genotype determination of 116 clinical isolates of A. fumigatus obtained from three healthcare centres. A new, automated and highly discriminatory typing method is described for A. fumigatus strains. The optimized multiplex PCR was successfully performed with all tested clinical strains and showed a discriminatory power of 0.9997 among presumably unrelated isolates. The comparison of groups of strains from different health centres showed that 99.6% of the genotypic variation was present within groups. Strains with the same genotype were isolated from the same patient, sometimes recovered more than 1 year later. A few cases of patients at the same clinic unit carrying strains of identical genotype strongly suggested colonization by A. fumigatus during their hospitalization. Specific measures must therefore be taken in order to prevent and restrict such incidents.

Susceptibility to five antifungals of Aspergillus fumigatus strains isolated from chronically colonised cystic fibrosis patients receiving azole therapy

Exposure of Aspergillus fumigatus to stressful antifungal therapies may result in decreased susceptibility. The aim of the present work was to evaluate the susceptibility to azole and non-azole antifungals of 159 isolates of A. fumigatus collected from cystic fibrosis (CF) patients receiving azole antifungal therapy. The genetic diversity of the fungal isolates was assessed using microsatellite genotyping, and some strains were found in patients sputum samples more than 4 years apart. No resistant isolates [minimal inhibitory concentration (MIC)/minimal effective concentration (MEC)>or=4 microg/mL] were identified to the antifungals amphotericin B, caspofungin, itraconazole and voriconazole. A single A. fumigatus isolate was identified outside of the epidemiological cut-off of 0.25 microg/mL for posaconazole. Susceptibility of the recurrent isolates was in agreement with the susceptibility of the first isolate identified (100% essential agreement). Even after azole exposure, several recurrent A. fumigatus strains were detected in the subsequent sputum samples. Development of resistance in A. fumigatus to antifungals appears to be rare amongst CF patients. However, it remains crucial to evaluate the importance of antifungal agents for allergic fungal diseases.

Genetic diversity of Aspergillus fumigatus in indoor hospital environments

Environmental isolates of Aspergillus fumigatus are less studied than those recovered from clinical sources. In the present study, the genetic diversity among such environmental isolates was assessed, as well as their dispersion ability and the acquisition of new strains in 19 medical units of the same hospital. A. fumigatus isolates were genotyped using a single multiplex PCR-based reaction with eight microsatellite markers and an insertion/deletion polymorphism. A total of 130 unique genotypes were found among a total of 250 A. fumigatus isolates. Genotypic diversity ranged from 0.86 to 1 in samples from hospital rooms, and there was no correlation between these samples and the presence of high-efficiency particulate air filters or any other air filtration system. Four of the six most prevalent A. fumigatus strains were recovered from water samples. The occurrence of microvariation was common among environmental isolates, which affected each of the microsatellite markers. The assessment of the genetic diversity of A. fumigatus is a useful tool for illustrating the presence or absence of specific clonal populations in a clinical setting. A. fumigatus populations were highly dynamic indoors, and new populations were found in just a few months. Due to the high indoor dispersion capability of A. fumigatus, more attention should be given to strains with increased pathogenic potential or reduced susceptibility to anti-fungal drugs.

Fungal infections after haematology unit renovation: evidence of clinical, environmental and economical impact

Objective and methods:  The Haemato‐Oncology Unit, Hospital S. Joao, suffered extensive refurbishing intervention in order to adapt for autotransplant patients. Eight new individual rooms with central HEPA filtration system were built. All patients admitted in the department during 14 months prior to and 14 months after renovation works were enrolled. A total of 403 admissions were considered and a detailed analysis of all patients with fungal infections, air quality and antifungal consumption were evaluated in order to study clinical, environmental and economical impact after unit renovation.

Interindividual variability and intraindividual stability of oral fungal microbiota over time

Oral microbiota is one of the most complex and diverse microbial communities in the human body. In the present study, we aimed to characterize oral fungi biodiversity and stability over time in a group of healthy participants with good oral health. Oral health and oral fungal microbiota were evaluated in 40 healthy individuals. A follow-up of 10 participants was carried out 28 weeks and 30 weeks after the first sampling. Oral rinse was collected and incubated in a fungal selective medium at 25ºC and 37ºC for 7 days. Fungi were identified based on macro- and microscopic morphology. API/ID32C was used for yeast identification, and molecular techniques were used to identify the most prevalent nonidentified moulds, mainly by sequencing 18S and internally transcribed spacer regions. Moulds were recovered from all participants and yeast from 92.5%. The most frequently isolated fungi were Candida spp., Rhodotorula spp., Penicillium spp., Aspergillus spp., and Cladosporium spp. The oral fungal community presented a high interindividual variability, but the frequency and quantification of each fungal taxon was constant over the 30-week observation period, showing a consistent intraindividual stability over time. The intraindividual stability opposed to interindividual variability may suggest a common and a variable group of fungi in the oral cavity.

Human albumin promotes germination, hyphal growth and antifungal resistance by Aspergillus fumigatus

Invasive aspergillosis is one of the most common deep-seated fungal infections among patients with an impaired immune system. Albumin is a serum protein commonly administered to critical patients. Our objective was to evaluate the in vitro effect of human albumin upon germination and hyphal growth of Aspergillus species, especially the most pathogenic, Aspergillus fumigatus, as well as its influence on antifungal drug activity. Human albumin induced, at normal serum concentrations (2-4%), a significant promotion of conidial germination by A. fumigatus, but not by Aspergillus flavus and Aspergillus niger. However, mycelium growth following germination was enhanced in all Aspergillus species. Minimal inhibitory concentrations (MIC) of all strains tested to amphotericin B and itraconazole increased in the presence of physiological concentrations of human albumin. Voriconazole activity was not, however, significantly affected by the presence of the protein. Conidial germination represents a crucial initial step in the progression to invasive disease, involving metabolic pathways that may differ considerably among Aspergillus species. Our results support the concept that human albumin may promote a faster onset and enhanced dissemination of invasive aspergillosis.

Susceptibility pattern among pathogenic species of Aspergillus to physical and chemical treatments

Physical treatments, like heating or irradiation, may reduce the viability or eradicate Aspergillus conidia, which in turn might help to prevent infections by members of this genus. Chemical treatments can also prevent infection resulting from contaminated hospital fabrics or surfaces. Our objectives were to study the kinetics of survival of the conidia of pathogenic Aspergillus species, like A. fumigatus, A. flavus and A. niger, during exposure to heating at 60 degrees C and microwave irradiation. In addition, we evaluated the susceptibility patterns of Aspergillus conidia to such chemical agents as cupric sulphate and sodium hypochlorite. Heating the conidia of A. flavus and A. niger at 60 degrees C for 45 min was found to be fungicidal (reduction > 104 conidia/ml), but was not with A. fumigatus conidia. Short periods of microwave irradiation (40 s) resulted in a significant reduction of the viability of the conidia of these three Aspergillus species as a result of lethal membrane lesions. All Aspergillus species were similarly susceptible to cupric sulphate and sodium hypochlorite. Therefore, heating, microwave and the chemical treatments tested impaired significantly the viability of Aspergillus conidia, supporting the use of these methods as preventive measures among patients at risk.

SNaPaer: A Practical Single Nucleotide Polymorphism Multiplex Assay for Genotyping of Pseudomonas aeruginosa

Multilocus sequence typing (MLST) represents the gold standard genotyping method in studies concerning microbial population structure, being particularly helpful in the detection of clonal relatedness. However, its applicability on large-scale genotyping is limited due to the high cost and time spent on the task. The selection of the most informative nucleotide positions simplifies genomic characterization of bacteria. A simple and informative multiplex, SNaPaer assay, was developed and genotyping of Pseudomonas aeruginosa was obtained after a single reaction of multiplex PCR amplification and mini-sequencing. This cost-effective technique allowed the analysis of a Portuguese set of isolates (n = 111) collected from three distinct hospitals and the genotyping data could be obtained in less than six hours. Point mutations were shown to be the most frequent event responsible for diversification of the Portuguese population sample. The Portuguese isolates corroborated the epidemic hypothesis for P. aeruginosa population. SNaPaer genotyping assay provided a discriminatory power of 0.9993 for P. aeruginosa, by testing in silico several hundreds of MLST profiles available online. The newly proposed assay targets less than 0.01% of the total MLST length and guarantees reproducibility, unambiguous analysis and the possibility of comparing and transferring data between different laboratories. The plasticity of the method still supports the addition of extra molecular markers targeting specific purposes/populations. SNaPaer can be of great value to clinical laboratories by facilitating routine genotyping of P. aeruginosa.