TABLE 1 - uploaded by Thomas J Walsh
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Diagnosis of invasive pulmonary aspergillosis (IPA) remains a major challenge to clinical microbiology laboratories. We developed
rapid and sensitive quantitative PCR (qPCR) assays for genus- and species-specific identification of Aspergillus infections by use of TaqMan technology. In order to validate these assays and understand their potential di...
Citations
... The diagnosis of invasive aspergillosis (IA) remains challenging for clinical microbiology laboratories (Walsh et al., 2011). Among human pathogenic Aspergillus species, A. fumigatus is the primary causative agent, followed by A. flavus, A. terreus, A. niger, and A. nidulans (Denning, 1998;Morgan et al., 2005;Dagenais and Keller, 2009). ...
The challenge of discriminating closely related species persists, notably within clinical diagnostic laboratories for invasive aspergillosis (IA)-related species and food contamination microorganisms with toxin-producing potential. We employed Analysis of the whole-GEnome (AGE) to address the challenges of closely related species within the genus Aspergillus and developed a rapid detection method. First, reliable whole genome data for 77 Aspergillus species were downloaded from the database, and through bioinformatic analysis, specific targets for each species were identified. Subsequently, sequencing was employed to validate these specific targets. Additionally, we developed an on-site detection method targeting a specific target using a genome editing system. Our results indicate that AGE has successfully achieved reliable identification of all IA-related species (Aspergillus fumigatus, Aspergillus niger, Aspergillus nidulans, Aspergillus flavus, and Aspergillus terreus) and three well-known species (A. flavus, Aspergillus parasiticus, and Aspergillus oryzae) within the Aspergillus section. Flavi and AGE have provided species-level-specific targets for 77 species within the genus Aspergillus. Based on these reference targets, the sequencing results targeting specific targets substantiate the efficacy of distinguishing the focal species from its closely related species. Notably, the amalgamation of room-temperature amplification and genome editing techniques demonstrates the capacity for rapid and accurate identification of genomic DNA samples at a concentration as low as 0.1 ng/μl within a concise 30-min timeframe. Importantly, this methodology circumvents the reliance on large specialized instrumentation by presenting a singular tube operational modality and allowing for visualized result assessment. These advancements aptly meet the exigencies of on-site detection requirements for the specified species, facilitating prompt diagnosis and food quality monitoring. Moreover, as an identification method based on species-specific genomic sequences, AGE shows promising potential as an effective tool for epidemiological research and species classification.
... For validation of A. fumigatus levels in mock and clinical respiratory samples, a TaqMan probe assay targeting the ITS1 region was used [39]. Assays were performed with TaqMan Fast Advanced Master Mix (ThermoFisher Scientific). ...
... The optimised method of the assay was applied to 10 clinical samples previously assessed by a clinically validated Aspergillus fumigatus qPCR test targeting the 18S rRNA gene. As samples had been in storage for a prolonged period (> 12 months), they were re-validated in-house to quantify the presence of A. fumigatus using a TaqMan qPCR assay targeting the ITS1 region [39]. Using a Ct cutoff of 40, 2 samples were negative, 3 were deemed positive at a level of < 1 GE and 5 at a level > 1 GE (Table 1). ...
Background
Amplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target.
Methods
Novel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed.
Results
The Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data.
Conclusions
This study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.
... After Aspergillus species, mucormycetes are the second most common cause of mycosis in hematological patients (1,2). Therefore, finding a fast and accurate method to identify mucor species is very important, especially in its clinical samples (3,4). For this reason, the use of new, fast, and sensitive methods, including molecular methods of deoxyribonucleic acid (DNA) detection in various samples, such as serum, BAL, and tissue, is currently being investigated (5,6). ...
Background: Mucormycosis is an invasive fungal infection that occurs in immunodeficiency patients and belongs to infections caused by mucoral fungi, such as Rhizopus and Rhizomucor. Objectives: This study investigated the identification and detection of the mucor fungus in tissue samples and other samples by the real-time polymerase chain reaction (PCR) method. Methods: This was a cross-sectional-descriptive study. A total of 80 tissue samples were collected from referring patients to diagnose fungi with the opinion of a specialist doctor. After extracting deoxyribonucleic acid (DNA) from the samples, PCR and real-time PCR tests were performed using specific primers for mucor and universal-pan and compared to culture. The results were confirmed by sequencing. Results: In this study, 80 samples were examined. In the PCR method, 74 and 75 cases of fungi were confirmed from the DNA obtained from the colonies and tissues, respectively. By using a specific primer for mucor, 12 patients were reported as positive for mucor. Additionally, 76 and 77 cases of fungi were confirmed in the real-time PCR method from the DNA obtained from the colonies and tissues, respectively, and positive cases were reported. By using specific primers and the real-time PCR test for mucor, 16 cases of mucor were reported from colonies, and 15 cases of mucor were reported positive from tissues. Conclusions: This study showed that real-time PCR and PCR are high-speed and more accurate methods than the culture test in identifying its species and have acceptable results in clinical cases.
... Aspergillosis in breeding ducks is a chronic disease that decreases productivity and causes economic losses (Zamboni et al, 2020). Among Aspergillus spp., mainly three species cause fungal lesions (Walsh et al, 2011). A. fumigatus is the most common cause of invasive pulmonary aspergillosis (IPA). ...
... A. fumigatus is the most common cause of invasive pulmonary aspergillosis (IPA). A. flavus is associated with the occurrence of combined sinopulmonary disease and nosocomial aspergillosis (Walsh et al, 2011). A. terreus is the cause of pulmonary and disseminated Aspergillus spp. ...
... Early diagnosis of fungal diseases and initiation of antifungal therapy based on the identification of the causative agent constitutes an effective treatment strategy (Walsh et al, 2011). ...
... The DNA concentration was determined using a Qubit 2.0 (Invitrogen, Waltham, MA, USA) fluorometer. Quantitative real-time PCR (qPCR) analysis was performed with CFX96 (BioRad, Vienna, Austria) using an iTaq Universal Probes supermix (BioRad) in a total volume of 20 µL with the primers and a probe (Supplementary Table S1), complementary to the ITS1 region of A. fumigatus [41] and to the oprL gene of P. aeruginosa [42] under the cycling parameters outlined in Supplementary Table S1. For the qPCR assay (performed in triplicates), the reaction mixture consisted of 10 µL of 2 × iTaq Supermix, 5 µL template DNA, and each primer (900 nM and 300 nM) for A. fumigatus and P. aeruginosa, respectively. ...
Pseudomonas aeruginosa is recognized as a significant cause of morbidity and mortality among nosocomial pathogens. In respiratory infections, P. aeruginosa acts not only as a single player but also collaborates with the opportunistic fungal pathogen Aspergillus fumigatus. This study introduced a QS molecule portfolio as a potential new biomarker that affects the secretion of virulence factors and biofilm formation. The quantitative levels of QS molecules, including 3-o-C12-HSL, 3-o-C8-HSL, C4-HSL, C6-HSL, HHQ, PQS, and PYO, measured using mass spectrometry in a monoculture, indicated metabolic changes during the transition from planktonic to sessile cells. In the co-cultures with A. fumigatus, the profile of abundant QS molecules was reduced to 3-o-C12-HSL, C4-HSL, PQS, and PYO. A decrease in C4-HSL by 50% to 170.6 ± 11.8 ng/mL and an increase 3-o-C12-HSL by 30% up to 784.4 ± 0.6 ng/mL were detected at the stage of the coverage of the hyphae with bacteria. Using scanning electron microscopy, we showed the morphological stages of the P. aeruginosa biofilm, such as cell aggregates, maturated biofilm, and cell dispersion. qPCR quantification of the genome equivalents of both microorganisms suggested that they exhibited an interplay strategy rather than antagonism. This is the first study demonstrating the quantitative growth-dependent appearance of QS molecule secretion in a monoculture of P. aeruginosa and a co-culture with A. fumigatus.
... In addition, Aspergillus species are a significant cause of opportunistic mycoses and invasive mycosis (6,7). Identifying Aspergillus species is critical because it provides vital information to guide the initiation of antifungal therapy (8)(9)(10) and offers clues about the disease spectrum. ...
This study addresses the challenge of accurately identifying filamentous fungi in medical laboratories using transfer learning with convolutional neural networks (CNNs). The study uses microscopic images from touch-tape slides with lactophenol cotton blue staining, the most common method in clinical settings, to classify fungal genera and identify Aspergillus species. The training and test data sets included 4,108 images with representative microscopic morphology for each genus, and a soft attention mechanism was incorporated to enhance classification accuracy. As a result, the study achieved an overall classification accuracy of 94.9% for four frequently encountered genera and 84.5% for Aspergillus species. One of the distinct features is the involvement of medical technologists in developing a model that seamlessly integrates into routine workflows. In addition, the study highlights the potential of merging advanced technology with medical laboratory practices to diagnose filamentous fungi accurately and efficiently.
IMPORTANCE This study utilizes transfer learning with CNNs to classify fungal genera and identify Aspergillus species using microscopic images from touch-tape preparation and lactophenol cotton blue staining. The training and test data sets included 4,108 images with representative microscopic morphology for each genus, and a soft attention mechanism was incorporated to enhance classification accuracy. As a result, the study achieved an overall classification accuracy of 94.9% for four frequently encountered genera and 84.5% for Aspergillus species. One of the distinct features is the involvement of medical technologists in developing a model that seamlessly integrates into routine workflows. In addition, the study highlights the potential of merging advanced technology with medical laboratory practices to diagnose filamentous fungi accurately and efficiently.
... The conventional polymerase chain reaction (PCR) technique for the identification of Aspergillus fumigatus was performed in lung and air sac samples from adult capercaillie 3, following a published protocol [24]. Briefly, the PCR was performed with 5 µM of each primer (5 -GCCCGCCGTTTCGAC-3 and 5 -CCGTTGTTGAAAGTTTTAACTGATTAC-3 ) for the specific amplification of A. fumigatus ITS1 region, using the DNA AmpliTools Multiplex Master Mix (Biotools, Madrid, Spain) in a GenAmp PCR System 9700 (Thermofisher Scientific, Barcelona, Spain). ...
The Cantabrian capercaillie (Tetrao urogallus cantabricus) is one of the most severely threatened subspecies of capercaillie. Its current population range is restricted to a small area of the Cantabrian Mountains (northwestern Spain), with only around 200 individuals remaining. As part of the national strategy for the conservation of the subspecies, the Cantabrian capercaillie Captive Breeding Center of Sobrescobio opened in 2009. Here, we use the information provided by the necropsies performed in this facility on 29 individuals (11 males, 13 females and 5 undetermined; 16 chicks and 13 adults) in order to describe the main mortality causes of captive-bred Cantabrian capercaillies. After necropsy, tissue samples were taken for evaluation using standard methods in histology and microbiology. The majority of the captive animals (18/29, 62.07%) died due to infectious diseases, mainly due to Escherichia coli, Clostridium perfringens, or Aspergillus fumigatus infection. The remaining 11 animals died due to stress-related processes (i.e., rupture of the heart apex and cardiomyopathy or neurogenic shock) (8/29, 27.59%), duodenal obstruction and coelomitis (1/29, 3.45%), perforation of the proventriculus and heart with a briar branch (1/29, 3.45%) or euthanasia due to a valgus leg deformity that prevented proper animal welfare (1/29, 3.45%). Young animals (i.e., younger than 2 months) died mainly due to infectious diseases (14/16, 87.5%), while stress-related causes were responsible for most adult deaths (7/13, 53.85%). We additionally report that two free-ranging adult males died due to exertional myopathy. This study provides relevant information for reducing mortality in captive capercaillies and improving both living conditions in captivity and the adaptation of these animals to the wild.
... In recent years, considerable efforts have been made to develop more sensitive and specific tools and protocols for IMD diagnosis. It is reported that PCR-based techniques, including conventional, semi-nested and real-time PCR, can be used to identify fungal agents in FFPE tissue (Bialek et al., 2005;Rickerts et al., 2006;Walsh et al., 2011;Springer et al., 2016a). RQ-PCR is very suitable for detecting the DNA of FFPE samples which are easily degraded. ...
... RQ-PCR is very suitable for detecting the DNA of FFPE samples which are easily degraded. There are reports using the 18SrRNA gene and the 28SrRNA gene regions to detect and distinguish mucormycosis and invasive aspergillosis (Bialek et al., 2005;Walsh et al., 2011;Springer et al., 2016a;Gade et al., 2017). ...
... Mucorales RQ-PCR primers and probes targeting the 18SrRNA gene and the 28SrRNA gene were described by Springer et al. (Springer et al. 2016a). Aspergillus RQ-PCR primers and probes targeting the 18SrRNA gene were described by Walsh et al. (Walsh et al., 2011). The protocols of RQ-PCR amplifications were performed as described previously (Walsh et al., 2011;Springer et al. 2016a) with the exception of the design of a new primer pair. ...
Background
In this study, we used real-time quantitative PCR (RQ-PCR) to rapidly detect Mucorales and Aspergillus in formalin-fixed, paraffin-embedded (FFPE) samples, targeting 18SrRNA gene and 28SrRNA gene. Identification of Mucorales and Aspergillus was analysed by combining Mucorales RQ-PCR (Mucorales18SrRNA and Mucorales28SrRNA) with Aspergillus RQ-PCR (Aspergillus18SrRNA and Aspergillus28SrRNA).
Objectives
The aims of this study were to compare the diagnostic performances of four RQ-PCR assays as single and combined diagnostic and identification tools.
Methods
We collected 12 control group samples and 81 experimental group samples diagnosed by histopathology, including mucormycosis (19 patients, 21 FFPE samples), aspergillosis (54 patients, 57 FFPE samples) and mucormycosis with aspergillosis (3 patients, 3 FFPE samples). All samples were detected by four RQ-PCR tests to compare and analyze diagnostic performance.
Results
The sensitivities of Mucorales18SrRNA and Mucorales28SrRNA were both 75%, with the tests having specificities of 97.10% and 94.20%. The sensitivities of Aspergillus18SrRNA and Aspergillus28SrRNA were 73.33% and 65%, with the tests having specificities of 87.88% and 81.82%. The values of the evaluation indexes of the combined detection of Mucorales28SrRNA and Aspergillus18SrRNA (M28A18) were the highest with a kappa coefficient value of 0.353, followed by M18A18. M28A18 had a sensitivity of 67.90% and a specificity of 100%.
Conclusions
We recommend using the combination of Mucorales RQ-PCR and Aspergillus RQ-PCR as a screening tool to detect samples suspected of mucormycosis and/or aspergillosis.
... The observation that the pan-A s p e r g i l l u s p r i m e r s a n d p r o b e cross-amplified Penicillium spp, but not Candida spp, was consistent with the report of Walsh et al (2011), owing to the close phylogenetic relationship between Penicillium and Aspergillus spp (Hope et al, 2005;Walsh et al, 2011). In the current study, two false-positive qPCR samples were due to Penicillium infection (data not shown), although Penicillium infections in humans are extremely rare (Lyratzopoulos et al, 2002). ...
... The observation that the pan-A s p e r g i l l u s p r i m e r s a n d p r o b e cross-amplified Penicillium spp, but not Candida spp, was consistent with the report of Walsh et al (2011), owing to the close phylogenetic relationship between Penicillium and Aspergillus spp (Hope et al, 2005;Walsh et al, 2011). In the current study, two false-positive qPCR samples were due to Penicillium infection (data not shown), although Penicillium infections in humans are extremely rare (Lyratzopoulos et al, 2002). ...
Microscopic detection of infectious fungus is frequently insensitive while traditional culture-based methods have limited sensitivity and specificity. PCR-based techniques provide rapid, sensitive and specific detection of pathogens. Quantitative (q)PCR, in comparison to direct microscopic examination and culture method, was carried out to identify Aspergillus flavus, A. fumigatus and A. niger, three of the most medically important pathogenic Aspergillus spp in clinical specimens from Hezhou People's Hospital in Guangxi province, PR China. Among clinical specimens (n = 161) collected between March and November 2020, qPCR-positive samples were obtained from female (n = 22) and male (n = 34) patients, with a median age of 61.0 and 62.5 years respectively (range = 10 months-92 years old). Excellent agreement was observed between the qPCR assay and culture technique (concordance rate Ƙ = 0.944, p-value <0.0001), while smear method demonstrated moderate agreement (concordance rate Ƙ = 0.476, p-value <0.0001). A. fumigatus, A. niger and A. flavus was identified in 75, 16 and 9%, respectively of positive specimens (n = 56) obtained from patients (n = 31) >60 years of age. Of 53 qPCR Aspergillus spp-positive specimens, 77, 9 and 9% were from sputum, ear secretion and abscess, respectively. The superior sensitivity, specificity and rapidity of qPCR compared to microscopic and culture techniques in the detection of Aspergillus spp in clinical samples despite its initial hardware cost.
... Most relevant species outside the Fumigati section are A. flavus, A. nidulans, A. terreus and A. niger [8]. A. fumigatus is the most frequently involved in human diseases in reports from America and Europe, while A. flavus is gaining prevalence in some Asian countries, notably in rhino-orbito-cerebral infections [9]. In patients' care, daily practice requires the identification of the strain to the level of section, whereas further identification, to the species level, is possible through the use of molecular testing [10]. ...
... Culture should be incubated at 30 °C for 21 days in a humidified environment. Mass spectrometry identification (MALDI-TOF) or genomic sequencing can be used on positive culture if microscopy does not enable a species identification [10,116]. Even more importantly, a positive culture enables testing of the strain's sensitivity to antifungals. ...
Invasive pulmonary aspergillosis is growing in incidence, as patients at risk are growing in diversity. Outside the classical context of neutropenia, new risk factors are emerging or newly identified, such as new anticancer drugs, viral pneumonias and hepatic dysfunctions. Clinical signs remain unspecific in these populations and the diagnostic work-up has considerably expanded. Computed tomography is key to assess the pulmonary lesions of aspergillosis, whose various features must be acknowledged. Positron-emission tomography can bring additional information for diagnosis and follow-up. The mycological argument for diagnosis is rarely fully conclusive, as biopsy from a sterile site is challenging in most clinical contexts. In patients with a risk and suggestive radiological findings, probable invasive aspergillosis is diagnosed through blood and bronchoalveolar lavage fluid samples by detecting galactomannan or DNA, or by direct microscopy and culture for the latter. Diagnosis is considered possible with mold infection in lack of mycological criterion. Nevertheless, the therapeutic decision should not be hindered by these research-oriented categories, that have been completed by better adapted ones in specific settings. Survival has been improved over the past decades with the development of relevant antifungals, including lipid formulations of amphotericin B and new azoles. New antifungals, including first-in-class molecules, are awaited.
