Population genomics confirms acquisition of drug-resistant Aspergillus fumigatus infection by humans from the environment.

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections.

Pulmonary Aspergillosis in Patients with Suspected Ventilator-associated Pneumonia in UK ICUs.

Rationale:Aspergillus infection in patients with suspected ventilator-associated pneumonia remains uncharacterized because of the absence of a disease definition and limited access to sensitive diagnostic tests.Objectives: To estimate the prevalence and outcomes of Aspergillus infection in adults with suspected ventilator-associated pneumonia.Methods: Two prospective UK studies recruited 360 critically ill adults with new or worsening alveolar shadowing on chest X-ray and clinical/hematological parameters supporting suspected ventilator-associated pneumonia. Stored serum and BAL fluid were available from 194 nonneutropenic patients and underwent mycological testing. Patients were categorized as having probable Aspergillus infection using a definition comprising clinical, radiological, and mycological criteria. Mycological criteria included positive histology or microscopy, positive BAL fluid culture, galactomannan optical index of 1 or more in BAL fluid or 0.5 or more in serum.Measurements and Main Results: Of 194 patients evaluated, 24 met the definition of probable Aspergillus infection, giving an estimated prevalence of 12.4% (95% confidence interval, 8.1-17.8). All 24 patients had positive galactomannan in serum (n = 4), BAL fluid (n = 16), or both (n = 4); three patients cultured Aspergillus sp. in BAL fluid. Patients with probable Aspergillus infection had a significantly longer median duration of critical care stay (25.5 vs. 15.5 d, P = 0.02). ICU mortality was numerically higher in this group, although this was not statistically significant (33.3% vs. 22.8%; P = 0.23).Conclusions: The estimated prevalence for probable Aspergillus infection in this geographically dispersed multicenter UK cohort indicates that this condition should be considered when investigating patients with suspected ventilator-associated pneumonia, including patient groups not previously recognized to be at high risk of aspergillosis.

A Comparison of Aspergillus and Mucorales PCR Testing of Different Bronchoalveolar Lavage Fluid Fractions from Patients with Suspected Invasive Pulmonary Fungal Disease.

In patients with hematological malignancies, bronchoalveolar lavage fluid (BALF) specimens are commonly used for the diagnosis of mold infections. However, it is not clear whether the cell pellet (P) or the supernatant fraction (S) of the BALF specimen is optimal for molecular diagnostic testing. Thus, 99 BALF specimens were collected from 96 hematology patients with or without allogeneic hematopoietic stem cell transplant. The cell pellets and supernatants were processed alone and in combination (S/P) for testing by two fungus-specific real-time PCR assays compliant with international recommendations. The results achieved with S/P were revealed to be superior in comparison to those achieved with S and P alone, with the use of each single fraction showing a reduced sensitivity for the detection of Aspergillus DNA (82% and 43% for S and P, respectively). In 57% of the samples, testing of the combination of S and P generated a lower quantification cycle value than testing of S or P alone. Molds would have been missed in 5 and 16 out of 28 samples if only S or P, respectively, was analyzed. No sample was positive by testing of S or P only. Similar results were obtained for the detection of Mucorales DNA in BALF specimens (reduced sensitivity of 67% and 50% for S and P, respectively). Study patients were categorized according to the current European Organization for the Research and Treatment of Cancer/Mycoses Study Group classification for invasive fungal disease (IFD), revealing that 35 patients had proven/probable IFD (36%), 47 patients had possible IFD (49%), and 14 patients had undetermined IFD (15%).

An evaluation of the performance of the Dynamiker® Fungus (1-3)-ß-D-Glucan Assay to assist in the diagnosis of Pneumocystis pneumonia.

The Dynamiker® Fungus (1-3)-ß-D-Glucan Assay (D-BDG) has recently become available in the Western Hemisphere for the diagnosis of invasive fungal disease (IFD). Evaluations of its performance for Pneumocystis pneumonia (PcP) are limited. A retrospective evaluation of D-BDG diagnosis of PcP was performed (23 PcP cases and 23 controls). Sensitivity and specificity were 87% and 70%, respectively, reducing the positivity threshold to 45 pg/ml increased sensitivity (96%), whereas a threshold of 300 pg/ml increased specificity (96%). The performance of D-BDG for the detection of PcP is comparable to other IFD, but sensitivity is below that required to confidently exclude PcP.

Analytical and Clinical Evaluation of the PathoNostics AsperGenius Assay for Detection of Invasive Aspergillosis and Resistance to Azole Antifungal Drugs Directly from Plasma Samples.

With the proposal to include Aspergillus PCR in the revised European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) definitions for fungal disease, commercially manufactured assays may be required to provide standardization and accessibility. The PathoNostics AsperGenius assay represents one such test that has the ability to detect a range of Aspergillus species as well as azole resistance in Aspergillus fumigatus Its performance has been validated on bronchoalveolar lavage (BAL) fluid and serum specimens, but recent evidence suggests that testing of plasma may have enhanced sensitivity over that with serum. We decided to evaluate the analytical and clinical performances of the PathoNostics AsperGenius assay for testing of plasma. For the analytical evaluations, plasma was spiked with various concentrations of Aspergillus genomic DNA before extraction following international recommendations, using two automated platforms. For the clinical study, 211 samples from 10 proven/probable invasive aspergillosis (IA) and 2 possible IA cases and 27 controls were tested. The limits of detection for testing of DNA extracted using the bioMérieux EasyMag and Qiagen EZ1 extractors were 5 and 10 genomes/0.5-ml sample, respectively. In the clinical study, true positivity was significantly greater than false positivity (P < 0.0001). The sensitivity and specificity obtained using a single positive result as significant were 80% and 77.8%, respectively. If multiple samples were required to be positive, specificity was increased to 100%, albeit sensitivity was reduced to 50%. The AsperGenius assay provided good clinical performance, but the predicted improvement of testing with plasma was not seen, possibly as a result of target degradation attributed to sample storage. Prospective testing is required to determine the clinical utility of this assay, particularly for the diagnosis of azole-resistant disease.

An evaluation of the performance of the Dynamiker® Fungus (1-3)-ß-D-Glucan Assay to assist in the diagnosis of invasive aspergillosis, invasive candidiasis and Pneumocystis pneumonia.

Invasive fungal disease (IFD) can be caused by a range of pathogens. Conventional diagnosis has the capacity to detect most causes of IFD, but poor performance limits impact. The introduction of non-culture diagnostics, including the detection of (1-3)-ß-D-Glucan (BDG), has shown promising performance for the detection of IFD in variety of clinical settings. Recently, the Dynamiker® Fungus (1-3)-ß-D-Glucan assay (D-BDG) was released as an IFD diagnostic test. This article describes an evaluation of the D-BDG assay for the diagnosis of invasive aspergillosis (IA), invasive candidiasis (IC) and Pneumocystis pneumonia (PCP) across several high-risk patient cohorts and provides comparative data with the Associates of Cape Cod Fungitell® and BioRad Platelia™ Aspergillus Ag (GM) assays. There were 163 serum samples from 121 patients tested, from 21 probable IA cases, 28 proven IC cases, six probable PCP cases, one probable IFD case, 14 possible IFD cases and 64 control patients. For proven/probable IFD the mean BDG concentration was 209pg/ml, significantly greater than the control population (73pg/ml; P: <.0001). The sensitivity, specificity, and diagnostic odds ratio for proven/probable IFD was 81.4%, 78.1%, and 15.5, respectively. Significant BDG false positivity (9/13) was associated post abdominal surgery. D-BDG showed fair and good agreement with the Fungitell®, and GM assays, respectively. In conclusion, the D-BDG provides a useful adjunct test to aid the diagnosis of IFD, with technical flexibility that will assist laboratories processing low sample numbers. Further, large scale, prospective evaluation is required to confirm the clinical validity and determine clinical utility.

Analytical and Clinical Evaluation of the PathoNostics AsperGenius Assay for Detection of Invasive Aspergillosis and Resistance to Azole Antifungal Drugs during Testing of Serum Samples.

The commercially developed PathoNostics AsperGenius species assay is a multiplex real-time PCR capable of detecting aspergillosis and genetic markers associated with azole resistance. The assay is validated for testing bronchoalveolar lavage fluids, replacing the requirement for culture and benefiting patient management. Application of this assay to less invasive, easily obtainable samples (e.g., serum) might be advantageous. The aim of this study was to determine the analytical and clinical performance of the AsperGenius species and resistance assays for testing serum samples. For the analytical evaluations, serum samples were spiked with various concentrations of Aspergillus genomic DNA for extraction, following international recommendations. For the clinical study, 124 DNA extracts from 14 proven/probable invasive aspergillosis (IA) cases, 2 possible IA cases, and 33 controls were tested. The resistance assay was performed on Aspergillus fumigatus PCR-positive samples when a sufficient fungal burden was evident. The limits of detection of the species and resistance assays for A. fumigatus DNA were 10 and ≥75 genomes/sample, respectively. Nonreproducible detection at lower burdens was achievable for all markers. With a positivity threshold of 39 cycles, the sensitivity and specificity of the species assay were 78.6% and 100%, respectively. For 7 IA cases, at least one genetic region potentially associated with azole resistance was successfully amplified, although no resistance markers were detected in this small cohort. The AsperGenius assay provides good clinical performance with the added ability to detect azole resistance directly from noninvasive samples. While the available burden will limit application, it remains a significant advancement in the diagnosis and management of aspergillosis.