Interrogation of related clinical pan-azole-resistant Aspergillus fumigatus strains: G138C, Y431C, and G434C single nucleotide polymorphisms in cyp51A, upregulation of cyp51A, and integration and activation of transposon Atf1 in the cyp51A promoter.

Multiple Aspergillus fumigatus isolates from a patient with two aspergillomas complicating chronic pulmonary aspergillosis were pan-azole resistant. Microsatellite typing was identical for all isolates despite major phenotypic and some growth rate differences. Three different cyp51A mutations were found (G138C, Y431C, and G434C), of which the first two were demonstrated by heterologous expression in a hypersusceptible Saccharomyces cerevisiae strain to be at least partly responsible for elevated MICs. cyp51A and cyp51B gene duplication was excluded, but increased expression of cyp51A was demonstrated in three isolates selected for additional study (7-to 13-fold increases). In the isolate with the greatest cyp51A expression, an Aft1 transposon was found inserted 370 bp upstream of the start codon of the cyp51A gene, an integration location never previously demonstrated in Aspergillus. Two transcription start sites were identified at 49 and 136 bp upstream of the start codon. The role of the Aft1 transposon, if any, in modulating cyp51A expression remains to be established. Increased mRNA expression of the transporters AfuMDR1 and AfuMDR4 also was demonstrated in some isolates, which could contribute to azole resistance or simply represent a stress response. The diversity of confirmed and possible azole resistance mechanisms demonstrated in a single series of isogenic isolates is remarkable, indicating the ability of A. fumigatus to adapt in the clinical setting.

A novel technique for the reconstruction of infected full-thickness chest wall defects.

BACKGROUND: Chest wall resection and reconstruction can be performed with minimal mortality and excellent functional and cosmetic results using synthetic meshes, methylmethacrylate, or other substitutes. However, these techniques are less easily applicable if chest wall resections have to be performed for infections. METHODS: We report a novel technique for this purpose using a modified latissimus dorsi flap harvested in continuity with the thoracolumbar fascia. The vascularized fascia was sutured into the chest wall defect, providing a stable base for the muscular component of the flap. Three patients requiring large full-thickness resections of the anterolateral chest wall for chronic infections were treated accordingly, two presenting with chronic radionecrosis and osteomyelitis and one with chest wall invasion by pulmonary aspergillosis. RESULTS: There were no intraoperative or postoperative complications and immediate extubation was possible in all 3 patients without the need for postoperative ventilation or tracheotomy. Healing of the infected chest wall was observed in all 3 patients. Postoperative cinemagnetic resonance imaging revealed concordant movements of the replaced segments without evidence of paradoxical motion during inspiration and expiration. CONCLUSIONS: This technique is easy and safe. It allows a stable and satisfactory reconstruction after large anterolateral full-thickness chest wall resections of infected, previously irradiated tissues, using only well-vascularized autologous tissue.

Immunoproteomics for Serological Diagnosis of Hypersensitivity Pneumonitis Caused by Environmental Microorganisms.

Diagnosis of immunoallergenic pathologies due to microorganisms such as hypersensitivity pneumonitis includes detection of circulating specific antibodies. Detection of precipitins has classically been performed using immunoprecipitation techniques with crude antigenic extracts from microorganisms implicated as etiologic agents. However, these techniques lack standardization because of the different composition of fungal antigenic extracts from one batch to another. Therefore, there is high interest in developing standardized serological diagnostic methods using recombinant antigens. Immunoproteomics have proved to be useful for identifying the immunogenic proteins in several microorganisms linked to hypersensitivity pneumonitis. With this approach, the causative microorganisms are first isolated from the environment of patients. Then the proteins are separated by two-dimensional electrophoresis and revealed by Western blotting with sera of different patients suffering from the disease compared to sera of asymptomatic exposed controls. Immunoreactive proteins are identified by mass spectrometry. Identified immunoreactive proteins found to be specific markers for the disease could be subsequently produced as recombinant antigens using various expression systems to develop ELISA tests. Using recombinant antigens, standardized ELISA techniques can be developed, with sensitivity and specificity reaching 80% and 90%, respectively, and more if using a combination of several antigens. Immunoproteomics can be applied to any environmental microorganisms, with the aim of proposing panels of recombinant antigens able to improve the sensitivity and standardization of serologic diagnosis of hypersensitivity pneumonitis, but also other mold-induced allergic diseases such as allergic broncho pulmonary aspergillosis or asthma.

Aspergillus fumigatus from normal and condemned carcasses with airsacculitis in commercial poultry

Carcass inspection is important for the detection of certain diseases and for monitoring their prevalence in slaughterhouses. The objective of this study was to assess the occurrence of aspergillosis caused by Aspergillus fumigatus in commercial poultry, through mycological and histopathological diagnosis, and to verify the causal association between the aspergillosis diagnosis criteria and condemnation due to airsacculitis in broilers through a case-control study. The study was carried out with 380 samples. Lungs were collected from broilers that were condemned (95) or not condemned (285) due to airsacculitis directly from the slaughter line. Forty-six (12%) lung samples were positive for A. fumigatus in mycological culture. Among all samples, 177 (46.6%) presented histopathological alterations, with necrotic, fibrinous, heterophilic pneumonia; heterophilic pneumonia and lymphoid hyperplasia being the most frequent. Out of the 380 lungs analyzed, 65.2% (30) showed histopathological alterations and isolation of fungi. The statistical analysis (McNemar's chi-square test) indicated a significant association between the presence of histopathological lesions and the isolation of A. fumigatus. Mycological cultivation and histopathological diagnosis increase the probability of detecting pulmonary alterations in birds condemned by the Final Inspection System, which suggests that such diagnostic criteria can improve the assessment and condemnation of birds affected by airsacculitis.

Fungal infections in marrow transplant recipients under antifungal prophylaxis with fluconazole

Fungal infection is one of the most important causes of morbidity and mortality in bone marrow transplant (BMT) recipients. The growing incidence of these infections is related to several factors including prolonged granulocytopenia, use of broad-spectrum antibiotics, conditioning regimens, and use of immunosuppression to avoid graft-versus-host disease (GvHD). In the present series, we report five cases of invasive mold infections documented among 64 BMT recipients undergoing fluconazole antifungal prophylaxis: 1) A strain of Scedosporium prolificans was isolated from a skin lesion that developed on day +72 after BMT in a chronic myeloid leukemic patient. 2) Invasive pulmonary aspergillosis (Aspergillus fumigatus) was diagnosed on day +29 in a patient with a long period of hospitalization before being transplanted for severe aplastic anemia. 3) A tumoral lung lesion due to Rhizopus arrhizus (zygomycosis) was observed in a transplanted patient who presented severe chronic GvHD. 4) A tumoral lesion due to Aspergillus spp involving the 7th, 8th and 9th right ribs and local soft tissue was diagnosed in a BMT patient on day +110. 5) A patient with a history of Ph1-positive acute lymphocytic leukemia exhibited a cerebral lesion on day +477 after receiving a BMT during an episode of severe chronic GvHD. At that time, blood and spinal fluid cultures yielded Fusarium sp. Opportunistic infections due to fungi other than Candida spp are becoming a major problem among BMT patients receiving systemic antifungal prophylaxis with fluconazole.

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Functional Characterization of an Aspergillus fumigatus Calcium Transporter (PmcA) that Is Essential for Fungal Infection

Aspergillus fumigatus is a primary and opportunistic pathogen, as well as a major allergen, of mammals. The Ca+2-calcineurin pathway affects virulence, morphogenesis and antifungal drug action in A. fumigatus. Here, we investigated three components of the A. fumigatus Ca+2-calcineurin pathway, pmcA,-B, and -C, which encode calcium transporters. We demonstrated that CrzA can directly control the mRNA accumulation of the pmcA-C genes by binding to their promoter regions. CrzA-binding experiments suggested that the 5'-CACAGCCAC-3' and 5'-CCCTGCCCC-3' sequences upstream of pmcA and pmcC genes, respectively, are possible calcineurin-dependent response elements (CDREs)-like consensus motifs. Null mutants were constructed for pmcA and -beta and a conditional mutant for pmcC demonstrating pmcC is an essential gene. The Delta pmcA and Delta pmcB mutants were more sensitive to calcium and resistant to manganese and cyclosporin was able to modulate the sensitivity or resistance of these mutants to these salts, supporting the interaction between calcineurin and the function of these transporters. The pmcA-C genes have decreased mRNA abundance into the alveoli in the Delta calA and Delta crzA mutant strains. However, only the A. fumigatus Delta pmcA was avirulent in the murine model of invasive pulmonary aspergillosis.

Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus

Aspergillus fumigatus is a major opportunistic pathogen and allergen of mammals. Nutrient sensing and acquisition mechanisms, as well as the capability to cope with different stressing conditions, are essential for A. fumigatus virulence and survival in the mammalian host. This study characterized the A. fumigatus SebA transcription factor, which is the putative homologue of the factor encoded by Trichoderma atroviride seb1. The Delta sebA mutant demonstrated reduced growth in the presence of paraquat, hydrogen peroxide, CaCl2, and poor nutritional conditions, while viability associated with sebA was also affected by heat shock exposure. Accordingly, SebA:GFP (SebA:green fluorescent protein) was shown to accumulate in the nucleus upon exposure to oxidative stress and heat shock conditions. In addition, genes involved in either the oxidative stress or heat shock response had reduced transcription in the Delta sebA mutant. The A. fumigatus Delta sebA strain was attenuated in virulence in a murine model of invasive pulmonary aspergillosis. Furthermore, killing of the Delta sebA mutant by murine alveolar macrophages was increased compared to killing of the wild-type strain. A. fumigatus SebA plays a complex role, contributing to several stress tolerance pathways and growth under poor nutritional conditions, and seems to be integrated into different stress responses.

Burden of serious fungal infections in Tanzania.

The incidence and prevalence of fungal infections in Tanzania remains unknown. We assessed the annual burden in the general population and among populations at risk. Data were extracted from 2012 reports of the Tanzanian AIDS program, WHO, reports, Tanzanian census, and from a comprehensive PubMed search. We used modelling and HIV data to estimate the burdens of Pneumocystis jirovecii pneumonia (PCP), cryptococcal meningitis (CM) and candidiasis. Asthma, chronic obstructive pulmonary disease and tuberculosis data were used to estimate the burden of allergic bronchopulmonary aspergillosis (ABPA) and chronic pulmonary aspergillosis (CPA). Burdens of candidaemia and Candida peritonitis were derived from critical care and/or cancer patients' data. In 2012, Tanzania's population was 43.6 million (mainland) with 1 500 000 people reported to be HIV-infected. Estimated burden of fungal infections was: 4412 CM, 9600 PCP, 81 051 and 88 509 oral and oesophageal candidiasis cases respectively. There were 10 437 estimated posttuberculosis CPA cases, whereas candidaemia and Candida peritonitis cases were 2181 and 327 respectively. No reliable data exist on blastomycosis, mucormycosis or fungal keratitis. Over 3% of Tanzanians suffer from serious fungal infections annually, mostly related to HIV. Cryptococcosis and PCP are major causes of mycoses-related deaths. National surveillance of fungal infections is urgently needed.

International expert opinion on the management of infection caused by azole-resistant Aspergillus fumigatus

Acknowledgements We thank Gilead Sciences Europe Ltd., UK for financially supporting logistical aspects of this study. The logistics of the meeting were handled by Congress Care, Den Bosch, The Netherlands. The sponsor was not involved in the selection of the participants or procedures, or in the discussion, data collection, analysis or writing of the manuscript. The medical writer was financially supported by the Dutch Society for Medical Mycology and the Department of Medical Microbiology, Radboud University Medical Centre, Nijmegen, The Netherlands.

Cerebral aspergillosis due to Aspergillus fumigatus in AIDS patient: first culture - proven case reported in Brazil

Cerebral aspergillosis is a rare cause of brain expansive lesion in AIDS patients. We report the first culture-proven case of brain abscess due to Aspergillus fumigatus in a Brazilian AIDS patient. The patient, a 26 year-old male with human immunodeficiency virus (HIV) infection and history of pulmonary tuberculosis and cerebral toxoplasmosis, had fever, cough, dyspnea, and two episodes of seizures. The brain computerized tomography (CT) showed a bi-parietal and parasagittal hypodense lesion with peripheral enhancement, and significant mass effect. There was started anti-Toxoplasma treatment. Three weeks later, the patient presented mental confusion, and a new brain CT evidenced increase in the lesion. He underwent brain biopsy, draining 10 mL of purulent material. The direct mycological examination revealed septated and hyaline hyphae. There was started amphotericin B deoxycholate. The culture of the material demonstrated presence of the Aspergillus fumigatus. The following two months, the patient was submitted to three surgeries, with insertion of drainage catheter and administration of amphotericin B intralesional. Three months after hospital admission, his neurological condition suffered discrete changes. However, he died due to intrahospital pneumonia. Brain abscess caused by Aspergillus fumigatus must be considered in the differential diagnosis of the brain expansive lesions in AIDS patients in Brazil.

Aspergillus fumigatus fungus ball in hospitalized patients with chronic pulmonary disease: usefulness of double immunodiffusion test as a screening procedure

Double immunodiffusion (DID) was used as a screening test for the diagnosis of aspergillosis. Three hundred and fifty patients were tested, all of them referred from a specialized chest disease hospital and without a definitive etiological diagnosis. When DID was positive addtional information such as clinical history and radiographic findings were requested and also surgical specimens were obtained whenever possible. Specific precipitin hamds for Aspergillus fumigatus antigen were found in 29 (8.3%) of 350 patients sera. Nineteen (65.5%) of the 29 patients with positive serology were recognized as having a fungus ball by X-rays signs in 17 or by pathological examination in 2 or by both in 8 patients. This two-year prospective study has shown that pulmonary aspergillos is a considerable problem among patiens admitted to a Chest Diseases Hospital, especially in those with pulmonary cavities or bronchiectasis.

Review of CT features of saprophytic aspergillosis (Aspergilloma) and correlation with final diagnosis : P21

Purpose: 1. To review Ct features suggestive of saprophytic aspergillosis (aspergilloma) and to correlate them with the final pathological results. 2. To illustrate the wide range of differential diagnosis. Methods and materials: The electronic database of our department from 1995 to 2007 revealed CT reports of 48 patients that had been considered very suggestive of aspergilloma. Two radiologists with 6 and 12 years experience in thoracic radiology jointly reviewed the corresponding CT features including ancillary findings and the underlying lung diseases and correlated them with the final pathological diagnosis. Results: Forty patients could be included in the study (12 women, mean age 52), while in 8 patients there was no adequate clinical follow-up. In 17 patients the diagnosis "mycetoma" due to aspergillus fumigatus infection was confirmed, either by surgery, biopsy or bronchoscopy. In 23 patients, differential diagnoses were found, such as cavitating bronchial carcinoma (n = 7), bacterial abscess (n = 3), typical (n = 2) and atypical (n = 2) tuberculosis, as well as inflammatory changes due to mucoviscidosis (n = 1), Wegener's disease (n = 1) or chronic obstructive pulmonary disease (n = 3). Fibromyxoide hamartoma, lung infarction and bronchomucocele were responsible for the typical CT feature in one patient each. Conclusion: 1. The typical CT feature suggesting mycetoma is softtissue proliferation within a pre-existing wall-thickened lung cavity, oten even considered "pathognomonic". However, this diagnosis was finally confirmed by surgery or laboratory findings in less than 50% of patients only. 2. Since differential diagnoses are very large, not only including cavitating lung cancer and tuberculosis, the individual underlying lung disease needs strongly being taken into account often giving the best clue for the correct diagnosis.

Complex pulmonary aspergilloma treated by cavernostomy

Objective: To evaluate the effectiveness of cavernostomy in patients with complex fungal balls.Methods: We analyzed the medical records of patients undergoing cavernostomy between January 2005 and May 2013, evaluating: age, gender, preoperative signs and symptoms, predisposing disease, preoperative tests, location of the aspergilloma, etiologic agent, cavernostomy indication, postoperative outcome.Results: Ten patients were male. The mean age was 42.9 years (34-56). The most frequent symptom was repeated pulmonary bleeding. Cavernostomy was proposed for patients at high risk for lung resection. It was performed in 17 patients and all of them had pulmonary tuberculosis sequelae, with cavitations. The indication in all cases was hemoptysis and elimination of phlegm. The cavernostomies were performed in a single surgical procedure. In all 17 patients the cavity was left open after the withdrawal of the mycetoma. In all patients hemoptysis ceased immediately. Operative mortality was 9.5% (1).Conclusion: cavernostomy is an effective treatment alternative in patients at high risk. It may be useful in some patients with complex aspergilloma, irrespective of lung function or bilateral disease. It is technically easy, has low-risk, saves parenchyma, and may be performed in a single operative time.

Invasive aspergillosis: a severe infection in juvenile systemic lupus erythematosus patients

Infections are an important cause of morbidity and mortality in juvenile systemic lupus erythematosus (JSLE). Among them, invasive aspergillosis (IA), which is usually related to immunosuppressed patients, has been rarely reported in JSLE. From 1983 to 2011, 5604 patients were followed at our institution and 283 (5%) met the American College of Rheumatology (ACR) classification criteria for SLE. Six (2.1%) of our JSLE patients had IA. One of them was previously reported and five will be described herein. Four of them were female. The median age at JSLE diagnosis was 12 years (8-16) and the median interval between diagnosis of JSLE and IA was 6 months (1-38). All had pulmonary involvement and three of them had systemic involvement. The median Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) was 19 (7-22). Diagnosis of IA was performed by isolation of Aspergillus spp., two in bronchoalveolar lavage culture and by way of autopsy in the others. All of them were treated with corticosteroids and/or immunosuppressive drugs at IA diagnosis (azathioprine and/or intravenous cyclophosphamide). They all required treatment in the pediatric intensive care unit with mechanical ventilation and antifungal therapy (fluconazole, amphotericin B, itraconazole and/or voriconazole); nonetheless, none of them survived. In conclusion, this was the first report that evaluated the prevalence of IA in a large population of JSLE patients from a tertiary pediatric hospital, and clearly showed the severity of the outcome, especially in patients with active disease and treated with immunosuppressive agents. This study reinforces the importance of early diagnosis and treatment with certain antifungals, especially in critically ill patients. Lupus (2012) 21, 1011-1016.