Fumigatus DNA Research Articles

Detection of azole-resistant Aspergillus fumigatus in the environment from air, plant debris, compost, and soil.

Aspergillus fumigatus is a ubiquitous fungus, a saprophyte of plants, and an opportunistic pathogen of humans. Azole fungicides are used in agriculture to control plant pathogens, and azoles are also used as a first line of treatment for aspergillosis. The continued exposure of A. fumigatus to azoles in the environment has likely led to azole resistance in the clinic where infections result in high levels of mortality. Pan-azole resistance in environmental isolates is most often associated with tandem-repeat (TR) mutations containing 34 or 46 nucleotides in the cyp51A gene. Because the rapid detection of resistance is important for public health, PCR-based techniques have been developed to detect TR mutations in clinical samples. We are interested in identifying agricultural environments conducive to resistance development, but environmental surveillance of resistance has focused on labor-intensive isolation of the fungus followed by screening for resistance. Our goal was to develop assays for the rapid detection of pan-azole-resistant A. fumigatus directly from air, plants, compost, and soil samples. To accomplish this, we optimized DNA extractions for air filters, soil, compost, and plant debris and standardized two nested-PCR assays targeting the TR mutations. Sensitivity and specificity of the assays were tested using A. fumigatus DNA from wild type and TR-based resistant isolates and with soil and air filters spiked with conidia of the same isolates. The nested-PCR assays were sensitive to 5 fg and specific to A. fumigatus without cross-reaction with DNA from other soil microorganisms. Environmental samples from agricultural settings in Georgia, USA were sampled and tested. The TR46 allele was recovered from 30% of samples, including air, soil and plant debris samples from compost, hibiscus and hemp. These assays allow rapid surveillance of resistant isolates directly from environmental samples improving our identification of hotspots of azole-resistant A. fumigatus.

Aspergillus tracheobronchitis with Birt-Hogg-Dubè syndrome as a rare cause of chronic cough

BackgroundAspergillus tracheobronchitis (ATB) is confined as a condition of chronic superficial infection of tracheobronchial tree. Its diagnosis is difficult due to atypical manifestations and low detective rate of Aspergillus thus far.Case presentation:Herein, we presented a 45-year-old male patient with a sole chronic productive cough for five years referred to our cough specialist clinic. Chest high-resolution computed tomography showed multiple lung cysts predominantly located in the subpleural lesions and near the mediastinum. Neither bacteria nor fungi were identified by sputum culture. However, metagenomic next-generation sequencing in sputum detected Aspergillus fumigatus DNA. The genetic testing of whole blood suggested the germline mutation of the tumor suppressor gene folliculin, supporting a diagnosis of Birt-Hogg-Dubé (BHD) syndrome. His productive cough symptom significantly improved after receiving itraconazole treatment for 2 months. After discontinuation of antifungal treatment, there was no relapse for four months follow-up. A diagnosis of ATB with BHD syndrome was eventually established in this patient.ConclusionATB should be considered in any patient with prolonged unexplained productive cough. Next-generation sequencing technologies may be useful to identify ATB which is uncommon and easily ignored in clinical practice.

P474 The value of PCR-based azole resistance detection in invasive aspergillosis: A prospective multicenter study

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesPrompt detection of azole-resistant Aspergillus fumigatus will result in the timely start of active treatment and may improve the survival of invasive aspergillosis (IA). The use of a multiplex polymerase chain reaction (PCR) targeting Aspergillus species and fumigatus DNA as well as the two most prevalent azole resistance- associated mutations (RAMs) in the cyp51A gen (TR34/L98H and TR46/Y121F/T289A) could shorten the time to detect azole-resistant IA.MethodsIn a prospective study in 12 Dutch and Belgian centers, we evaluated the clinical value of the multiplex AsperGenius®PCR in hematology patients with a pulmonary infiltrate undergoing bronchoalveolar lavage (BALf) sampling. The primary endpoint was antifungal treatment failure in the 6 weeks after antifungal treatment initiation in the patients in which azole-resistant IA was detected. Treatment failure was defined as death or a switch to an antifungal agent from another class after at least 5 days of first-line therapy. Patients with a mixed azole-susceptible/resistant infection were excluded from this analysis to ascertain that the infection was indeed caused by the resistant strain.ResultsOf 323 patients enrolled, sufficient BALf for PCR testing remained in 299. Probable fungal disease was diagnosed in 95 (34%), Aspergillus cultured in 24 (8%), Aspergillus DNA detected in 118 (39%), and A. fumigatus DNA in 88 (29%) patients. The resistance PCR was conclusive in 54/88 (61%) and RAMs were detected in 8 (15%), Table 1. All 8 had probable IA but 2 had a mixed infection and were excluded. In the 6 remaining patients, treatment failure was observed in one. Compared with the GM negative patients and despite antifungal therapy, a positive GM test was associated with a 13% higher 6-week overall mortality (P = .01), Table 2. Surprisingly, the 6-week mortality in the 65 patients who had a positive Aspergillus PCR but a negative GM and culture was not increased compared to those with a negative PCR (PCR + 14% vs. PCR- 16% mortality, P = .68).ConclusionsIn patients with an underlying hematological disease and a pulmonary infiltrate, the detection of Aspergillus DNA by PCR on BALf was not associated with increased mortality. The exact place of the Aspergillus PCR in the EORTC-MSGERC invasive fungal infection criteria is therefore uncertain. In 15% of the patients in whom A. fumigatus DNA was present, azole RAMs were detected by PCR. In only 1/6 probable cases of IA with RAMs detected, antifungal treatment failure was observed. Basing the choice of antifungal therapy on the result of a cyp51a resistance PCR may help to reduce the impact of azole resistance on mortality.

P407 Standardization of PCR for the diagnosis of invasive aspergillosis and invasive mucormycosis from blood samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives(1) Standardization of PCR-based diagnosis of invasive aspergillosis and invasive mucormycosis in spiked blood samples. (2) Standardization of PCR-based diagnosis of invasive aspergillosis and invasive mucormycosis in proven cases. (3) Validation of the above-standardized protocol in patients with proven/probable invasive aspergillosis or mucormycosis.Materials and Methods: Blood samples were collected from healthy volunteers and patients with proven mucormycosis, proven invasive aspergillosis, and suspected invasive fungal infection. Standard strains of Mucorales and Aspergillus spp. were collected from the National Culture Collection of Pathogenic Fungi, PGIMER and DNA was extracted using a modified protocol of the fungal DNA extraction method by Lee and Taylor (1990). Mucor-specific primer (ZM13), described by Bialek R et al. (2005) was used for mucormycosis samples. Aspergillus specific primer pair was designed from mitochondrial DNA of A. fumigatus using Primer3 software. Genomic DNA was used to determine the optimum annealing temperature and concentration for ZM13 assay. DNA extracted from whole blood samples of healthy volunteers after spiking with a known quantity of A. fumigatus DNA and only genomic DNA was used separately to optimize annealing temperature and concentration of Aspergillus-specific primers. Similarly, whole blood, plasma, and serum samples from healthy volunteers were spiked with various concentrations of Rhizopus arrhizus and A. fumigatus DNA. DNA extracted from spiked samples was used to determine the analytical sensitivity of PCR assays. DNA extracted from blood samples of ten proven mucormycosis and five invasive aspergillosis patients were used to determine the optimum sample dilution factor and template volume for the assay. The final standardized protocols were validated using 28 proven/probable cases of invasive aspergillosis and invasive mucormycosis and thirty cases of suspected invasive fungal infection.ResultsThe optimum annealing temperature and primer concentrations for the assay were 54.3°C and 0.4μm respectively for ZM13 assay and 53°C and 0.45μm respectively for Aspergillus assay. The limit of detection was 0.06fg/μl for ZM13 assay and 0.08fg/μL for Aspergillus specific assay. For mucormycosis assay, the slope of the standard curve was −3.2687 and the percentage efficiency of the assay was 104% with 0.9936 as the coefficient of determination (R2). Likewise, the slope of the standard curve was −3.2695, the percentage efficiency of the assay 102%, and the coefficient of determination (R2) 0.9858 for aspergillosis assay. The best combination of template volume and sample dilution factor was 2.5 μl and 1000x in whole blood samples and, 2.5 μl and 100x in serum samples for mucormycosis assay. It was 1.0 μl and 10x in serum samples for Aspergillus specific assay. The sensitivity, specificity, positive predictive value, and negative predictive value of mucormycosis qPCR using serum samples were 79.3%, 86.4%, 65.7%, and 92.7% respectively. The assay had a positive likelihood ratio of 5.92 and a negative likelihood ratio of 0.24.ConclusionThe real-time PCR protocols standardized for the diagnosis of mucormycosis and invasive aspergillosis from blood samples showed promising results. However, the assay needs further validation in a larger study population.

Utility of fungal polymerase chain reaction on nasal swab samples in the diagnosis and monitoring of sinonasal aspergillosis in dogs

BackgroundIn dogs with sinonasal aspergillosis (SNA) the utility of PCR in the diagnosis and monitoring of the disease after treatment has not been assessed.ObjectivesTo evaluate the presence of fungal DNA using quantitative PCR targeting Aspergillus fumigatus (Aspfum) and Aspergillus spp. (PanAsp), and PCR targeting multiple fungal species (PanFun), in samples obtained from nasal cavities of dogs with SNA, other nasal diseases and healthy dogs.AnimalsSixty‐two dogs including 20 with SNA, 12 with cured SNA (of which 10 are from the SNA group), 20 dogs with Non‐SNA nasal disease, and 20 healthy dogs.MethodsProspective cross‐sectional study. Aspfum, PanAsp, and PanFun were performed on blindly collected nasal swabs obtained in anesthetized dogs.ResultsIn SNA dogs, Aspfum and PanAsp were positive in 13/20 and 14/20 dogs. In all dogs in the 3 other groups, A. fumigatus DNA was not detected using Aspfum. PanAsp was positive in 3 non‐SNA dogs: 1 with cured SNA and 2 with Non‐SNA nasal disease. A Ct cut‐off value of 33.3 for Aspfum demonstrated 65% sensitivity and 100% specificity. A Ct cut‐off value of 34.5 for PanAsp demonstrated 70% sensitivity and 96.2% specificity. PanFun was positive in 16/20, 12/12, 19/20, and 7/20 dogs in the SNA, cured SNA, Non‐SNA, and healthy groups, respectively.Conclusion and Clinical Importance Aspfum and PanAsp on blindly collected nasal swabs can be useful for the detection of SNA at diagnosis and at cure, especially when more invasive methods are not available.

Evaluation of Three Commercial PCR Assays for the Detection of Azole-Resistant Aspergillus fumigatus from Respiratory Samples of Immunocompromised Patients.

This is the first study comparing three commercially available PCR assays for the detection of Aspergillus DNA from respiratory specimen of immunocompromised patients and the presence of cyp51A gene mutations. Bronchoalveolar lavages (BALs, N = 103) from patients with haematological/oncological underlying diseases were retrospectively investigated. The performance of three PCR assays, namely MycoGENIE® Aspergillus fumigatus Real-Time PCR Kit (Adamtech), Fungiplex® Aspergillus Azole-R IVD Real-Time PCR Kit (Bruker Daltonik GmbH) and AsperGenius® (PathoNostics B.V.), were evaluated. All patients were categorised following current EORTC/MSG criteria, with exclusion of the PCR-results. From the 11 invasive pulmonary aspergillosis (IPA) probable samples, eight were detected with MycoGENIE®, resulting in a sensitivity of 80% and a specificity of 73%. Furthermore, Fungiplex® resulted in six positive BALs with a sensitivity of 60% and a specificity of 91% and AsperGenius® in seven positive BAL samples, with a sensitivity of 64% and a specificity of 97%. No proven IPA was detected. One isolate showed phenotypically an azole-resistance, which was also detected in each of the tested PCR assays with the mutation in TR34. The here tested PCR assays were capable of reliably detecting A. fumigatus DNA, as well as differentiation of the common cyp51A gene mutations. However, evaluation on the AsperGenius® assay revealed a low risk of false positive results.

Comparative Pathomorphological, Mycological and Molecular Examination of Turkey Poults with Different Immunological Status Experimentally Infected with Aspergillus fumigatus

Abstract The aim of this study was to determine the pathological, mycological and molecular findings in turkey poults with different immunological status experimentally infected with Aspergillus fumigatus. The investigation was carried out 1, 3, 7, 14 and 21 days after intratracheal inoculation of 5.056×107 spores of A. fumigatus to 14-day-old turkey poults in group G-1, as well as to turkey poults in group G-2 which were treated prior to infection with dexamethasone. A. fumigatus was isolated on day 1 p.i. in both groups, but the number of positive samples was bigger in group G-1. A. fumigatus was isolated from the respiratory organs of group G-1as early as on day 1 and 3 p.i. in 4 out of 12 examined specimens (33%). On day 7 p.i. A. fumigatus was possible to isolate from the respiratory organs of 50% of infected birds, on day 14 in 83.33% and on day 21 p.i. A. fumigatus was isolated in 6 out of 6 sacrificed turkey poults (100%). In dexamethasone-treated group A. fumigatus isolates from the respiratory organs on day 1 and 3 p.i. were same as in group G-1, whereas on days 7 and 14 p.i. the number of turkey poults positive to A. fumigatus increased in comparison with the untreated G-1 group. The histopathological lesions in turkey poults treated with dexamethasone developed earlier, were more intensive and extensive. The mycological and nested PCR results revealed a higher number of samples positive for the presence of A. fumigatus DNA in the group G-2, pretreated with dexamethasone.

Evaluation of Two Commercial Real-Time PCR Kits for Aspergillus DNA Detection in Bronchoalveolar Lavage Fluid in Patients with Invasive Pulmonary Aspergillosis

Invasive pulmonary aspergillosis (IPA) is a common complication of immunosuppression. Rapid diagnosis using molecular techniques is essential to improve patient survival. PCR techniques are promising in enhancing Aspergillus detection in blood and respiratory samples. We evaluate for the first time the performances of two commercial real-time PCR kits, the A. fumigatus Bio-Evolution and the MycoGENIE A. fumigatus for the detection of A. fumigatus DNA in bronchoalveolar lavage (BAL) from patients with and without IPA. Seventy-three BAL samples were included. Thirty-one of them corresponded to patients with probable IPA, 11 to patients with possible IPA, and 31 to patients without aspergillosis, according to the 2008 European Organization for Research and Treatment of Cancer/Mycoses Study Group criteria. In the probable IPA group, A. fumigatus Bio-Evolution and the MycoGENIE A. fumigatus real-time PCR kits showed a specificity of 100% and a sensitivity of 81% and 71%, respectively. The A. fumigatus Bio-Evolution detected Aspergillus DNA in the 14 BAL samples with a positive Aspergillus culture result, whereas the MycoGENIE A. fumigatus PCR result was positive only for 12. In the possible IPA group, there were no positive real-time PCR or positive Aspergillus culture results. For the patients without aspergillosis, no positive result was observed for real-time PCR kit, despite the presence of various other non-Aspergillus pathogens in this group. Our study demonstrates an excellent specificity and a good sensitivity of A. fumigatus DNA detection in BAL samples with both kits.

Circulating Aspergillus fumigatus DNA Is Quantitatively Correlated to Galactomannan in Serum.

The performance of antigen galactomannan (GM) for diagnosing invasive aspergillosis (IA) is hampered by the occurrence of false-positive results. Quantitative PCR has been proposed to improve the diagnosis of IA. Therefore, we analyzed the value of performing a PCR test to the GM-positive serum sample. Using a quantitative PCR assay specific for Aspergillus fumigatus 28S ribosomal DNA, we retrospectively tested 422 GM-positive (Platelia Bio-Rad kit) serum samples collected over 1 year from 147 patients. The cases were classified based on EORTC criteria as “proven,” “probable,” and “no–IA” before availability of the PCR results. After exclusion of 65 samples for non-reproducibility of GM positivity (n = 62) or PCR inhibition (n = 3), 75 (21.0%) of the remaining 357 samples were PCR-positive. GM and fungal DNA showed a significantly positive correlation (p < 0.0001, R2 = 0.27, slope = 0.98 ± 0.19). At least one PCR-positive result was observed in 63.3% (31/49) of IA patients and in 13.2% (13/98) of non-IA patients (p < 0.0001). The PCR positivity was also associated with the presence of other microbiological criteria among the 44 patients with IA and complete mycological workup (p = 0.014), as well as a higher mortality rate at six months among the 135 patients with hematological conditions (p = 0.0198). Overall, we found a quantitative correlation between serum GM and circulating DNA with an increased likelihood of IA when both were positive. A PCR-positive result also supported a higher fungal load when GM was already positive. We advocate adding a PCR test for every confirmed GM-positive serum sample.

A prospective study of fungal biomarkers to improve management of invasive fungal diseases in a mixed specialty critical care unit

PurposeThe diagnosis of invasive fungal diseases (IFD) in critical care patients (CrCP) is difficult. The study investigated the performance of a set of biomarkers for diagnosis of IFD in a mixed specialty critical care unit (CrCU). MethodsA prospective observational study in patients receiving critical care for ≥7days was performed. Serum samples were tested for the presence of: (1-3) - β-d-glucan (BDG), galactomannan (GM), and Aspergillus fumigatus DNA. GM antigen detection was also performed on bronchoalveolar lavage (BAL) samples. The patients were classified using published definitions for IFD and a diagnostic algorithm for invasive pulmonary aspergillosis. Performance parameters of the assays were determined. ResultsIn patients with proven and probable IFD, the sensitivity, specificity, PPV and NPV of a single positive BDG were 63%, 83%, 65% and 83% respectively. Specificity increased to 86% with 2 consecutive positive results. The mean BDG value of patients with proven and probable IFD was significantly higher compared to those with fungal colonization and no IFD (p value<0.0001). ConclusionNew diagnostic criteria which incorporate these biomarkers, in particular BDG, and host factors unique to critical care patients should enhance diagnosis of IFD and positively impact antifungal stewardship programs.

Performance evaluation of multiplex PCR including Aspergillus—not so simple!: Table 1.

Multiplex PCRs have been designed for including species other than Aspergillus fumigatus for the diagnosis of invasive aspergillosis, such as microarrays, liquid-phase array, and electrospray-ionization mass spectrometry (PCR/ESI MS). These methods are based on the selection of multiple primers to amplify different species with the specificity checked by hybridization to a probe or by base composition of the amplicon for the PCR/ESI MS. When testing complex samples such as respiratory specimens, some clinically relevant species can be missed. Indeed, it is impossible to design primers able to amplify all the known fungal species with the same efficiency. Therefore, the best amplified species may not be the most clinically relevant. Multiplex assays have also been proposed to detect A. fumigatus DNA and azole resistance. Since the gene responsible for azole resistance is single copy and the gene used for detection is multicopy, only the high fungal loads can be evaluated. Thus, although interesting for investigating mycobiome, the multiplex assays should be used with cautious for the diagnosis of IA or the detection of resistance. For the diagnosis of invasive aspergillosis, validated quantitative PCRs specifically targeting A. fumigatus or a limited set of species to increase sensitivity is a safer option.

Determination of azole antifungal drug resistance mechanisms involving Cyp51A gene in clinical isolates of Aspergillus fumigatus and Aspergillus niger

Aims: The main aim of this research is to investigate azole resistance mechanisms in A. fumigatus and A. niger which involve Cyp51A gene that encodes 14-α sterol demethylase enzyme. Methodology and Results: Itraconazole susceptibility was determined through E-test method. A conventional PCR method was used to amplify and sequence Cyp51A gene in fungal DNA, to detect the presence of gene mutations. Real-time PCR method was applied to determine overexpression of Cyp51A gene in A. fumigatus and A. niger isolates. Susceptibility test found that 3/13 (23.1%) A. fumigatus and 7/23 (30.4%) A. niger isolates were resistant to Itraconazole, with minimum inhibitory concentrations (MICs) of 2.5 μg/ml to 3.0 μg/ml. Sequencing of A. fumigatus DNA showed presence of L98H mutation in 7/13 (53.8%) and M220 mutation in 3/13 (23%) isolates. Whereas, sequencing of A. niger DNA detected the presence of G427S mutation in 3/23 (13%) isolates. Tandem Repeat mutation was not detected in all A. fumigatus and A. niger isolates. Only M220 mutation showed significant correlation (r (13)=0.041038, p<0.05) with Itraconazole antifungal resistance in A. fumigatus isolates while L98H mutation was not involved. G427S mutation also showed correlation (r (15)=0.038434, p<0.05) with Itraconazole antifungal resistance in A. niger isolates. A higher level of Cyp51A gene expression was detected in 4/8 (50%) A. fumigatus isolates and 7/12 (58.3%) A. niger isolates. Resistant isolates more often showed higher level of Cyp51A gene expression compared to susceptible isolates; however the difference in level of expression between resistant isolates and susceptible isolates is not significant. This may be due to similar MIC values in resistant and susceptible isolates. Conclusion, significance and impact of study: In conclusion the level of azole resistance in A. fumigatus and A. niger isolates in Malaysia is low and mutations in Cyp51A gene may contribute towards Itraconazole antifungal resistance, however other factors may also be involved.

Determination of Azole Antifungal Drug Resistance Mechanisms Involving Cyp51a Gene in Clinical Isolates of Aspergillus fumigatus and Aspergillus niger

Aims: The main aim of this research is to investigate azole resistance mechanisms in A. fumigatus and A. niger which involve Cyp51A gene that encodes 14-α sterol demethylase enzyme. Methodology and Results: Itraconazole susceptibility was determined through E-test method. A conventional PCR method was used to amplify and sequence Cyp51A gene in fungal DNA, to detect the presence of gene mutations. Real-time PCR method was applied to determine overexpression of Cyp51A gene in A. fumigatus and A. niger isolates. Susceptibility test found that 3/13 (23.1%) A. fumigatus and 7/23 (30.4%) A. niger isolates were resistant to Itraconazole, with minimum inhibitory concentrations (MICs) of 2.5 μg/ml to 3.0 μg/ml. Sequencing of A. fumigatus DNA showed presence of L98H mutation in 7/13 (53.8%) and M220 mutation in 3/13 (23%) isolates. Whereas, sequencing of A. niger DNA detected the presence of G427S mutation in 3/23 (13%) isolates. Tandem Repeat mutation was not detected in all A. fumigatus and A. niger isolates. Only M220 mutation showed significant correlation (r (13)=0.041038, p<0.05) with Itraconazole antifungal resistance in A. fumigatus isolates while L98H mutation was not involved. G427S mutation also showed correlation (r (15)=0.038434, p<0.05) with Itraconazole antifungal resistance in A. niger isolates. A higher level of Cyp51A gene expression was detected in 4/8 (50%) A. fumigatus isolates and 7/12 (58.3%) A. niger isolates. Resistant isolates more often showed higher level of Cyp51A gene expression compared to susceptible isolates; however the difference in level of expression between resistant isolates and susceptible isolates is not significant. This may be due to similar MIC values in resistant and susceptible isolates. Conclusion, significance and impact of study: In conclusion the level of azole resistance in A. fumigatus and A. niger isolates in Malaysia is low and mutations in Cyp51A gene may contribute towards Itraconazole antifungal resistance, however other factors may also be involved.

Evaluation of mold exposure in cystic fibrosis patients' dwellings and allergic bronchopulmonary risk

Very few studies have been conducted on cystic fibrosis (CF) patients' exposure to the indoor environment and, to our knowledge, there are no studies dealing with the link between specific fungal environmental exposure at home and fungal colonization resulting in allergic bronchopulmonary aspergillosis (ABPA). Fungal exposure of CF adult patients with ABPA (n=4) with fungal sensitization (n=7) and with no ABPA (n=5) was assessed in 16 homes by dust sampling with electrostatic dust fall collectors (EDCs). Aspergillus fumigatus was specifically quantified by real-time quantitative polymerase chain reactions (qPCRs), and A. fumigatus DNA concentrations were significantly higher in homes of ABPA patients (p<0.001). Results indicate that indoor fungal contamination could be a factor favoring ABPA and suggest that environmental surveys could help in preventing fungal risk in CF patients.

Efficacy of DNA amplification in tissue biopsy samples to improve the detection of invasive fungal disease

The performance of a pan-fungal PCR-based technique was evaluated to assess the aetiology of invasive fungal diseases (IFDs). A total of 89 formalin-fixed paraffin-embedded biopsy samples from 84 patients with proven IFD were studied. Culture of tissue was performed in 68 (81%) patients. The sensitivities of the PCR-based technique and microbiological culture of tissues were 89% and 56%, respectively (p <0.01). According to PCR results, Aspergillus species accounted for 67%, Candida species for 13%, zygomycetes for 11%, and rare and emerging fungi for 9%. Aspergillus species were significantly associated with lung samples (79.6%, p <0.01), Mucorales were associated with skin/subcutaneous samples, and Candida species were associated with gastrointestinal samples. Regarding biopsy samples with Aspergillus species, Aspergillus fumigatus DNA was detected in 43 of 50 (86%), and Aspergillus flavus in six of 50 (12%). PCR was positive in 24 of 30 (80%) cases with negative culture. In nine of the 84 patients, the PCR technique failed to amplify the DNA. Six also had negative cultures, and in the remaining three cases culture was positive (Rhizopus microsporus, Rhizopus arrhizus, and Sakseneae vasiformis), suggesting that the PCR technique was not as effective in amplifying the DNA of some species of Zygomycetes. In five cases, there was no correlation between culture results and those obtained with DNA amplification, indicating the possibility of a mixed infection or the presence of colonizer/contaminant microorganisms. In summary, PCR-based techniques for DNA amplification should be implemented in histopathology and microbiology departments, as they appear to be complementary to conventional methods for IFD detection.

The Effect Analysis of Different Experimental Methods for the Diagnosis of Invasive Pulmonary Aspergillosis in a Rat Model

Background: Consensus on the most reliable assays to detect invasive aspergillosis from minimally or noninvasive samples has not been reached. In this study, we compared the efficacy of an enzyme-linked immunosorbent assay (ELISA) for galactomannan (GM) detection and quantitative real-time PCR assay (qRT-PCR) for the diagnosis of invasive pulmonary aspergillosis in a rat model. Methods: Neutropenic, male Sprague-Dawley rats (specific pathogen free; 8 weeks old; weight, 200 ± 20 g) were immunosuppressed with cyclophosphamide and infected with Aspergillus fumigatus intratracheally. Tissue and whole blood samples were harvested on days 1, 3, 5, and 7 post-infection and examined with GM ELISA and qRT-PCR. Results: On day 7, A. fumigatus DNA was amplified from 14 of 48 whole blood samples from immunosuppressed infected rats: day 1 (0/12), day 3 (0/12), day 5 (6/12), day 7 (8/12) post infection. The sensitivity and specificity of the qRT-PCR assay were 29.2% and 100%, respectively. Receiver operating characteristic curve (ROC) analysis indicated a Ct cut-off value of 15.35, and the area under the curve (AUC) was 0.627. The GM assay detected antigen in sera obtained on day 1 (5/12), day 3 (9/12), day 5 (12/12), and day 7 (12/12) post-infection, and thus had a sensitivity of 79.2% and a specificity of 100%. The ROC of the GM assay indicated that the optimal cut-off value was 1.40 (specificity, 100%; AUC, 0.919). Conclusions: The GM assay was more sensitive than qRT-PCR assay in diagnosing invasive pulmonary aspergillosis in rats.

Development of Novel PCR Assays To Detect Azole Resistance-Mediating Mutations of theAspergillus fumigatus cyp51AGene in Primary Clinical Samples from Neutropenic Patients

The increasing incidence of azole resistance in Aspergillus fumigatus causing invasive aspergillosis (IA) in immunocompromised/hematological patients emphasizes the need to improve the detection of resistance-mediating cyp51A gene mutations from primary clinical samples, particularly as the diagnosis of invasive aspergillosis is rarely based on a positive culture yield in this group of patients. We generated primers from the unique sequence of the Aspergillus fumigatus cyp51A gene to establish PCR assays with consecutive DNA sequence analysis to detect and identify the A. fumigatus cyp51A tandem repeat (TR) mutation in the promoter region and the L98H and M220 alterations directly in clinical samples. After testing of the sensitivity and specificity of the assays using serially diluted A. fumigatus and human DNA, A. fumigatus cyp51A gene fragments of about 150 bp potentially carrying the mutations were amplified directly from primary clinical samples and subsequently DNA sequenced. The determined sensitivities of the PCR assays were 600 fg, 6 pg, and 4 pg of A. fumigatus DNA for the TR, L98H, and M220 mutations, respectively. There was no cross-reactivity with human genomic DNA detectable. Sequencing of the PCR amplicons for A. fumigatus wild-type DNA confirmed the cyp51A wild-type sequence, and PCR products from one azole-resistant A. fumigatus isolate showed the L98H and TR mutations. The second azole-resistant isolate revealed an M220T alteration. We consider our assay to be of high epidemiological and clinical relevance to detect azole resistance and to optimize antifungal therapy in patients with IA.

Molecular diagnosis of Aspergillus fumigatus endocarditis

A 66-year-old male with ischaemic cardiomyopathy and chronic lymphocytic leukemia developed signs of severe systemic inflammatory response syndrome. Serial blood cultures were negative and a SeptiFast test detected the presence of Aspergillus fumigatus DNA. Afterwards, detection of galactomannan and 1,3-β-d-glucan showed a positive result. Autopsy revealed the presence of branched fungal structures suggestive of Aspergillus.

Real-time PCR and quantitative culture for monitoring of experimental Aspergillus fumigatus intracranial infection in neutropenic mice

The central nervous system (CNS) is the most common site of dissemination during Aspergillus infection. PCR has the potential to facilitate early diagnosis of CNS aspergillosis, which could assist in reducing disease mortality. In two experiments, neutropenic CD-1 male mice were infected intracranially with 5×10⁶ conidia of Aspergillus fumigatus. At time points up to 120 h after infection, mice were euthanized and samples of blood, brain, spinal cord and cerebrospinal fluid (CSF) were taken. The brain fungal burden was determined by quantitative culture, and fungal DNA was detected by quantitative PCR. Plating for A. fumigatus from the brain confirmed that all mice had burdens of log₁₀>3 from 4 to 120 h after infection. A. fumigatus DNA was detected in blood (88 %), brain (96 %), CSF (52 %) and spinal cord (92 %) samples. The brain and spinal cord contained the highest concentrations of fungal DNA. Adapting the extraction protocol to maximize yield from small sample volumes (10 µl CSF or 200 µl blood) allowed PCR detection of A. fumigatus in infected mice, suggesting the use of CSF and blood as diagnostic clinical samples for CNS aspergillosis.

Modification of an expression vector for efficient recombinant production and purification of mitogillin of Aspergillus fumigatus expressed in Escherichia coli

The diagnostic potential of secretory proteins of Aspergillus fumigatus is limited by their availability in pure form. We have constructed a vector (pGES-PH-1) to express genes encoding secretory proteins of A. fumigatus as fusion proteins with glutathione S-transferase (GST) in Escherichia coli. The mitogillin, a secretary protein of A. fumigatus, was expressed and purified to homogeneity by using pGES-PH-1. Mitogillin gene was PCR amplified from A. fumigatus DNA, cloned in pGES-PH-1 and expressed in E. coli as fusion protein with GST at N-terminal and 6xHis tag at C-terminal end. Pure mitogillin was obtained by purification on glutathione–Sepharose, cleavage of column-bound fusion protein by PreScission protease and by further purification on Ni–NTA-agarose. Polyclonal anti-mitogillin antibodies were raised in rabbits and were used to study its secretion during in vitro growth of A. fumigatus. The mitogillin was detectable in culture filtrate after 24 h of A. fumigatus growth and thereafter its amount increased progressively until 96 h in both, Sabouraud dextrose broth and potato dextrose broth. However, the secretion of mitogillin in culture medium was slightly delayed when A. fumigatus was grown in a minimal medium as mitogillin was detected only after 36 h of growth. Our results demonstrate the utility of the newly constructed expression vector with two affinity tags and PreScission protease cleavage site for high-level expression and efficient purification of a recombinant A. fumigatus secretory protein expressed in E. coli, which could be used for further studies.