Aspergillus PCR: One Step Closer to Standardization
P. Lewis WhiteStéphane BretagneLena KlingsporWillem J. G. MelchersElaine McCullochBettina SchulzNiklas FinnströmCarlo MengoliRosemary A. BarnesJ. Peter DonnellyJuergen Loeffler
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ABSTRACT PCR has been used as an aid in the diagnosis of invasive aspergillosis for almost 2 decades. A lack of standardization has limited both its acceptance as a diagnostic tool and multicenter clinical evaluations, preventing its inclusion in disease-defining criteria. In 2006, the European Aspergillus PCR Initiative was formed. The aim of the initiative was to provide optimal standardized protocols for the widespread clinical evaluation of the Aspergillus PCR to determine its diagnostic role and allow inclusion in disease diagnosis criteria. Quality control panels were developed and circulated to centers for evaluation of the existing methodology before recommendations based on the initial results were proposed for further panels. The centers were anonymously classified as “compliant” or “noncompliant,” according to whether they had followed the proposed recommendations before the performance parameters were determined and meta-regression analysis was performed. Most PCR amplification systems provided similar detection thresholds, although positivity was a function of the fungal burden. When PCR amplification was combined with DNA extraction, 50% of the centers failed to achieve the same level of detection. Meta-regression analysis showed positive correlations between sensitivity and extraction protocols incorporating the proposed recommendations and the use of bead beating, white cell lysis buffer, and an internal control PCR. The use of elution volumes above 100 μl showed a negative correlation with sensitivity. The efficiency of the Aspergillus PCR is limited by the extraction procedure and not by PCR amplification. For PCR testing of whole blood, it is essential that large blood volumes (≥3 ml) be efficiently lysed before bead beating to disrupt the fungal cell and performance of an internal control PCR to exclude false negativity. DNA should be eluted in volumes of <100 μl.Aspergillus niger
Ornamental plant
Pulmonary aspergillosis
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When a new hospital opened in 1983, environmental culturing for Aspergillus organisms and surveillance for nosocomial aspergillosis cases were begun to characterize the relationship between environmental contamination and infection. Monthly air sampling demonstrated increasing concentrations of Aspergillus flavus and Aspergillus fumigatus to mean levels >1 cfu/m3 during 1986–1987, accompanied by a progressive increase in incidence ofaspergillosis to 1.2% in immunocompromised patients. This prompted an inspection that revealed heavy growth of Aspergillus organisms on air filters. Subsequent inspections of hospital wards showed small foci of A. flavus growth on other materials. Removal of the contaminated filters and improved environmental maintenance were associated with reduction in A. flavus and A. fumigatus to 0.01 cfu/m3 and a fourfold decline in aspergillosis incidence during the next 2 years. These findings, together with laboratory studies that showed aspergilli could proliferate on common hospital materials when moistened, indicate a need for careful environmental maintenance.
Replication
Pathogenic organism
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Invasive fungal diseases (IFD) are a life-threatening infectious complications in immunocompromised patients and are associated with high rate of morbidity and mortality. The most common invasive mycosis in patients who underwent an allogeneic hematopoietic stem cell transplantation is invasive aspergilosis (IA), most frequently caused by the clinically dominant species Aspergillus fumigatus and, rarely, also by Aspergillus flavus, Aspergillus terreus and Aspergillus niger. In recent years, other related Aspergillus species were also reported to cause IFD, phenotypically similar to A. fumigatus and moreover, frequently exhibiting resistance towards various antifungals. For example, it is Aspergillus lentulus, Aspergillus viridinutans, Neosartoya fischeri, etc. Classical microbiological methods such as direct microscopy or culture are usually used for the identification of Aspergillus species. The application of PCR-based molecular techniques and monitoring of secondary metabolites production enable detection and identification of species, which are not distinguishable solely by their morphology. PCR methods are also useful for molecular strain typing of aspergilli and can reveal the genetic diversity of isolates.
Aspergillus terreus
Aspergillus niger
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Aspergillus nidulans
Dozen
Aspergillus niger
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Objective To evaluate the incidence of Aspergillus airway colonization after liver transplantation and the risk of invasive aspergillosis after colonization.Method All culture and histologic specimens obtained from a consecutive series of 56 liver transplant cases were reviewed for the presence of Aspergillus and compared with clinical data.Result Aspergillus was isolated from the airway in 24 of 56 transplant recipients (43%),invasive aspergillosis occurred in 2 cases with Aspergillus fumigatus colonized within the first 6 months of posttransplant and was uniformly fatal,account for 29% (2/7) of all posttransplant deaths. No patients developed invasive aspergillosis without Aspergillus colonization.Conclusion Aspergillus airway colonization after liver transplantation is common and in most cases,transient,In contrast,invasive aspergillus infection is less common but fatal.Invasive aspergillosis occurred only in patients initially colonized with A.fumigatus within the first 6 months of posttransplant,empiric anti-Aspergillus therapy during the first 6 months posttransplant may be warranted.
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Invasive aspergillosis is a common infection in patients who are immunocompromised. The diagnosis of invasive aspergillosis is difficult in the absence of confirmation by tissue biopsy and histological studies. Therefore, recent advances that may be important for the development of highly sensitive and specific serodiagnostic tests for the early diagnosis of invasive aspergillosis are reviewed. The inability of the detection of antibody to Aspergillus to lead to early diagnosis of invasive aspergillosis also is emphasized. However, sensitive methods that reliably detect significant amounts of aspergillus antigen in body fluids of high-risk patients are currently being evaluated and may provide a noninvasive early diagnostic test that is both sensitive and specific. Also, current antifungal agents with anti-aspergillus activity that have potential as therapeutic or prophylactic agents are reviewed briefly.
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Air sampling of the rooms and corridors of the oncology wards of the hospital was carried out over a 54-week period to assess the concentration of viable Aspergillus conidia. A. fumigatus and A. flavus were recovered at a mean of 1.83 cfu m-3 air sampled. Individual samplings yielded concentrations of up to 11.6 cfu m-3. Other Aspergillus spp. were recovered at a mean of 2.38 cfu m-3 (maximum 32.6 cfu m-3). Concentration was not correlated with season or hospital ward. Review of autopsy results showed an average of 6.6 cases of aspergillosis annually over a 22-year period. No seasonal variation in case incidence was found. Six cases of invasive aspergillosis were diagnosed on the three cancer wards during the air-sampling period, but no association was seen linking these cases with changes in recovery of airborne Aspergillus. A seasonal pattern was not observed in the overall incidence of aspergillosis cases nor concentrations of airborne conidia.
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Aspergillus is a ubiquitous mould genus typically found in soil and rotting vegetation. Sources, modes and treatment. In defining the diseases caused by Aspergillus, the term' aspergillosis 'is used but most generally refers to those caused by Aspergillus fumigatus. Aspergillus flavus, Aspergillus terreus and Aspergillus niger are other animals that can cause human illness [1]. Aspergillus releases massive numbers of conidia (asexual spores) into the air as part of its life cycle and can thus be present in both outdoor and indoor environments. Aspergillus conidia inhalation is normally a daily phenomenon, but only a limited number of individuals experience chronic illness and are at an elevated risk of aspergillosis (e.g. people with compromised immune systems and/or impaired lungs). It is difficult to quantify the burden of aspergillosis in the UK because of the insensitivity of fungal culture, the lack of regular, sensitive, non-culture diagnostic testing and the lack of a national surveillance network. A 2017 study estimated that 3,288-4,257 cases of invasive aspergillosis, up to 3,600 cases of recurrent pulmonary aspergillosis and 110,667-235,070 cases of allergic bronchopulmonary aspergillosis (ABPA) complicating asthma or cystic fibrosis are registered every year in the UK [2].
Allergic bronchopulmonary aspergillosis
Aspergillus terreus
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