Screening of cryptic species among clinical Aspergillus isolates collected during one year period in a Portuguese reference laboratory

Objectives: Correct identification of Aspergillus species is important given that sibling species may show variable susceptibilities to multiple antifungal drugs and also because sharper definition of species may facilitate epidemiological studies. Thus, we screened Aspergillus clinical isolates from Portuguese hospitals to determine which, if any, of the cryptic species of Aspergillus were involved in patient infections. Methods: Over a one year period, Aspergillus isolates from Portuguese health institutions were collected. These isolates were identified on the basis of microscopic morphology and through the use of molecular tools. Genomic DNA was prepared from each isolate and the sequencing of the Internal Transcribed Spacers (ITS) regions, specifically the ITS1 and ITS2 non-coding regions flanking the 5.8S rDNA was used to determine the species complex, whereas β-tubulin and calmodulin sequencing was done to achieve the correct species identification. Results: Over the study period, 57 Aspergillus isolates from clinical samples were collected from 10 Portuguese health institutions. According to the morphological observations, 29 isolates were identified as Aspergillus fumigatus, 11 A. flavus, 8 A. niger, 3 A. nidulans, 2 A. terreus, 2 A. candidus and 2 Aspergillus sp. Among those isolates, six species-complexes were detected by ITS sequencing, and were distributed as follows: fumigati (50.1%), flavi (21.0%), nigri (15.8%), terrei (5.3%), nidulantes (3.6%) and versicolores (3.6%). β-tubulin and calmodulin sequencing resulted in ten (17.5%) cryptic species being identified among the 57 isolates. Six of those isolates belonged to the nigri complex (A. brasiliensis, A. awamorii and A. tubigensis), two to the versicolores complex (A. sidowii and A. fructus), one to the fumigati complex (A. lentulus) and one to the nidulantes complex (Emmericella echinulata). Conclusion: With rigorous application of molecular tools, cryptic species of Aspergillus are not uncommon in the clinic. The identification of cryptic species among the collected clinical isolates of Aspergillus alerts the clinician to isolates with reduced susceptibilities to antifungal drugs and emphasizes a correct identification to species level

Surveillance of environmental fungi, with focus on Aspergillus, in a Portuguese Central Hospital.

Objectives: Because immunocompromised patients are more prone to acquire nosocomial infections caused by fungi isolated from the environment, e.g. Aspergillus, this study aimed to screen the hospital environment for the presence of fungi and to understand their epidemiology in the different hospital wards analyzed. Methods: During one-year period, four seasonal samplings, i.e., air and hard surface, were performed. A total of 101 air samples and 99 surface samples were collected from the Hematology, Oncology, and Intensive Care Unit (ICU) wards of a Portuguese Central Hospital. Aspergillus isolates were plated for growth as single colonies on malt extract agar with chloramphenicol to check the colony purity and observe colonial morphology. The universal fungal primers ITS1 and ITS4 were used to amplify DNA from all Aspergillus isolates, amplimers were sequenced, and isolates identified to the species-complex level. Statistical analyses were done using SPSS v15.0 program for Windows. Results: Aspergillus was the most frequently recovered fungal genus (20.9%), followed by Cladosporium (18.7%), and Penicillium (17.2%). Thirty-five Aspergillus isolates were collected from the wards with hematological patients (bone marrow transplant and hemato-oncology wards), whereas 15 isolates were recovered from ICU. Among Aspergillus isolates from the hospital environment, those belonging to the species-complexes of versicolores (n = 26; 32.5%), nigri (n = 12; 15.0%), flavi (n = 11; 13.7%), and circumdati (n = 6; 7.5%) dominated. Hemato-Oncology was the ward with higher fungal counts, whereas the bone marrow transplant ward, which is protected by HEPA-filtration of the supply air, showed the lowest numbers in all sampling periods. A significant association (p = 0.001) was found between the season and the Aspergillus complexes isolated, with spring and summer having a larger number of different species-complexes detected in the hospital´s air and on the surfaces. Nevertheless, air counts showed that the autumn was the season with the highest proportion of Aspergillus (one third of the total number of fungi detected). This could be due in part to the presence of construction work near these wards. Conclusion: The knowledge of the epidemiology of environmental fungi in each hospital may allow the establishment of preventive or corrective measures to decrease nosocomial fungal infections

Identification of Aspergillus cryptic species in hospital environment

Selected hospital wards, housing patients at higher risk to develop invasive fungal infections, were screened in order to understand the epidemiology and distribution of Aspergillus, especially regarding the presence of cryptic species.Aspergillus species were identified by b-tubulin and calmodulin sequencing, and a high percentage of cryptic species (i.e., not sensu stricto) was found (59%). Sections Usti, Versicolores and Circumdati harbored the highest proportion of cryptic species [100% (4/4), 95% (19/20) and 90% (9/10), respectively].The high number of cryptic species found raises concerns about the possible reduced susceptibility to antifungals of hospital environmental Aspergillus isolates. These data reinforce the importance of hospital air and surface monitoring, mainly in immunocompromised patients’ wards

Molecular screening of 246 Portuguese Aspergillus isolates among different clinical and environmental sources

This article was awarded the Young Investigator Prize, 6th Trends in Medical Mycology, Copenhagen, 2013.Clinical and environmental samples from Portugal were screened for the presence of Aspergillus and the distribution of the different species-complexes determined to understand differences in their distribution from different sources. Fifty-seven Aspergillus isolates from clinical samples were collected from 10 health institutions. Six species-complexes were detected by ITS sequencing: Fumigati, Flavi, and Nigri were the most frequent (50.9%, 21.0% and 15.8%, respectively). β-tubulin and calmodulin sequencing resulted in seven cryptic species (A. awamorii, A. brasiliensis, A. fructus, A. lentulus, A. sydowii, A. tubigensis, and Emmericella echinulata) being identified among the 57 isolates. Thirty-nine isolates of Aspergillus were recovered from beach sand and poultries, 31 from swineries, and 80 from hospital environments, for a total 189 isolates. Eleven species-complexes were found in these 189 isolates, and those belonging to the Versicolores species-complex were found the most frequently (23.8%). There was a significant association between the different environmental sources and the distribution of the several species-complexes; the hospital environment had greater variability of species-complexes than other environmental locations. A high prevalence of cryptic species within Circumdati complex was detected in several environments and from the isolates analyzed, at least four cryptic species were identified, most of them growing at 37ºC. Because Aspergillus species-complexes have different susceptibilities to antifungals, knowing the species-complex epidemiology for each setting, as well as the identification of cryptic species among the collected clinical isolates is important. This may allow preventive or corrective measures to be taken, which may result in decreased exposure to those organisms and a better prognosis.R. S. was financially supported by a fellowship from Fundação para a Ciência e Tecnologia Portugal (contract SFRH/BPD/72775/2010)

Deteção de espécies crípticas de Aspergillus com suscetibilidade reduzida a antifúngicos em ambiente hospitalar

Resumo da comunicação publicado em: Saúde em Números Portugal.Invasive aspergillosis is a fungal infection caused by Aspergillus spp. affecting mainly the immunocompromised. The mortality rate may reach 85%. Aspergillus identification should be based on molecular methods as there are species morphologically similar but distinct at the molecular level (cryptic species), with variable antifungal susceptibility profiles

Molecular Epidemiology of Aspergillus collected from Cystic Fibrosis Patients

Background: Aspergillus respiratory infection is a common complication in cystic fibrosis (CF) and is associated with loss of pulmonary function and allergic disease. Methods: Fifty-three Aspergillus isolates recovered fromCF patients were identified to species by Internal Transcribed Spacer Region (ITS), β-tubulin, and calmodulin sequencing. Results: Three species complexes (Terrei, Nigri, and Fumigati) were found. Identification to species level gave a single Aspergillus terreus sensu stricto, one Aspergillus niger sensu stricto and 51 Aspergillus fumigatus sensu stricto isolates. No cryptic species were found. Conclusions: To our knowledge, this is the first prospective study of Aspergillus species in CF using molecular methods. The paucity of non-A. fumigatus and of cryptic species of A. fumigatus suggests a special association of A. fumigatus sensu stricto with CF airways, indicating it likely displays unique characteristics making it suitable for chronic residence in that milieu. These findings could refine an epidemiologic and therapeutic approach geared to this pathogenRaquel Sabino was financially supported by a fellowship from Fundação para a Ciência e Tecnologia (FCT) Portugal (contract SFRH/BPD/72775/2010). This study was partially supported by a grant from the Child Health Research Institute, Stanford University, and a gift from Mr. John Flatley. José A.G. Ferreira was partially supported by a grant from the Brazilian National Council for Scientific and Technological Development (CNPq)

Recombinant cytokines in the therapy of fungal infection

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Amphotericin B concentrations in healthy mallard ducks (Anas platyrhynchos) following a single intratracheal dose of liposomal amphotericin B using an atomizer

Item does not contain fulltextAspergillosis is a fungal infection that primarily affects the respiratory tract. Amphotericin B has broad antifungal activity and is commonly used to treat aspergillosis, a fungal pneumonia that is a common sequela in oiled waterfowl as well as other birds in wildlife rehabilitation. Pharmacokinetic parameters of nebulized amphotericin B in an avian model have been reported, but those of direct intratracheal delivery have yet to be established. The objective of this study was to evaluate if a single 3 mg/kg dose of liposomal amphotericin B delivered intratracheally using a commercial atomizer would achieve plasma and lung tissue concentrations exceeding targeted minimum inhibitory concentrations (MIC) for Aspergillus species in adult mallard ducks (Anas platyrhynchos). Following intratracheal delivery, amphotericin B was present in lung parenchyma at concentrations above the targeted MIC of 1 mug/g for up to 9 days post-administration; however, distribution of the drug was uneven, with the majority of the drug concentrated in one lung lobe. Concentrations in the contralateral lung lobe and the kidneys were above the targeted MIC 1 day after administration but declined exponentially with a half-life of approximately 2 days. Plasma concentrations were never above the targeted MIC. Histological examination of the trachea, bronchi, lungs, heart, liver, and kidneys did not reveal any toxic changes. Using a commercial atomizer, intratracheal delivery of amphotericin B at 3 mg/kg resulted in lung parenchyma concentrations above 1 mug/ml with no discernable systemic effects. Further studies to establish a system of drug delivery to both sides of the pulmonary parenchyma need to be performed, and the efficacy of this treatment for disease prevention remains to be determined