Fumigatus Complex Research Articles

NCMP-29. BRAIN LESIONS IN A TEEN WITH NEWLY DIAGNOSED T-CELL ALL AND SARS-COV-2 INFECTION

Abstract T-cell Lymphoblastic Leukemia (T-cell ALL) accounts for up to 15% of all newly diagnosed cases of acute lymphoblastic leukemia in pediatric patients (0-18 years old). Patients are at risk of opportunistic infections during induction therapy with up to 2% developing blood stream infections when their absolute neutrophil count is above 1,500. We report a case of a 15-year-old female with High-Risk T-cell ALL, CNS1 (negative CNS disease), presenting with three days of headaches, photophobia, and non-neutropenic fever. She completed induction chemotherapy one week earlier with vincristine, daunorubicin, calaspargase and a 28-day course of dexamethasone. On physical exam, she had no focal deficits or changes in sensation or strength. CBC was notable for a white blood cell count: 7500, hemoglobin: 8.6, platelets: 328,000 and ANC (Absolute Neutrophil Count): 4700. The patient was also COVID positive on admission. A contrast enhanced MRI brain demonstrated multiple diffusion restricting and peripherally enhancing lesions, concerning for intracranial abscesses. Neurosurgery performed a burr hole craniotomy and aspirated 5 mL of purulent drainage, the culture of which grew Aspergillus fumigatus complex. The patient was started empirically on Voriconazole, in addition to broad spectrum antibiotic coverage pending culture results, due to excellent CNS penetration and proven superiority to amphotericin-based therapy for invasive aspergillosis. Micafungin was added due to CT chest demonstrating nodular infiltrates, suggesting primary pulmonary aspergillosis with CNS dissemination. The patient improved symptomatically without further sequelae. Patients with COVID-19 on prolonged courses of corticosteroids are at higher risk of developing fungal co-infections regardless of neutrophil count. While pulmonary aspergillosis is the most common location of a COVID-19 aspergillus coinfection, there have been documented reports of intracranial micro-abscesses. Contrast-enhanced brain MRI should be obtained in any patient with a recent history of COVID-19 presenting with new neurological symptoms, especially in a patient with a recent history of corticosteroids.

Whole-Genome Sequencing of Newly Emerged Fungal Pathogen Aspergillus Lentulus and Its Azole Resistance Gene Prediction.

Aims: Aspergillus lentulus is an important newly recorded species in the A. fumigatus complex and its resistance to azole drugs and the high mortality rate of infected individuals have emerged as problems. Comprehensive understanding of the A. lentulus is limited due to lack of genome-wide fine mapping data. The aim of this study was to investigate the A. lentulus signature at the molecular level, analyze the genome-wide profile of this strain, and predict its possible genes that execute azole resistance. Methods: In this study, a whole-genome sequencing of a clinically isolated A. lentulus strain (named A. lentulus PWCAL1) was studied by PacBio Sequel sequencing platform. Azole resistance genes were predicted based on whole-genome sequencing data analysis, gene function annotation, comparative genomic analysis, and BLASTP alignment using the Mycology Antifungal Resistance Database to comprehensively understanding the genome-wide features, pathogenicity, and resistance mechanisms of A. lentulus. Results: The results of whole-genome sequencing demonstrated that the total length of A. lentulus PWCAL1 genome was 31255105 bp, the GC content was 49.24%, and 6883 coding genes were predicted. A total of 4565, 1824, and 6405 genes were annotated in the Gene Ontology, Clusters of Orthologous Groups, and Kyoto Encyclopedia of Genes and Genomes databases, respectively. In the Pathogen Host Interactions Database and the Database of Fungal Virulence Factors, 949 and 259 interacting virulence factors were identified, respectively, with the main virulence factor-mutant virulence phenotype, being enriched in reduced virulence. Comparative genomic analysis showed that there were 5456 consensus core genes in this strain and four closely related strains of A. fumigatus complex, which were mainly involved in human diseases, metabolism, organismal systems, etc. Among the three aligned A. lentulus strains, the number of unique genes of this bacterium was the highest with a number of 171, and these genes were mainly associated with carbohydrate metabolism and cell growth and death. Resistance gene prediction demonstrated that the A5653 gene of this bacterium had F46Y/N248T double point mutations on the CYP51A gene, but no tandem repeat mutations in the promoter region were detected. Furthermore, 12 genes belonging to the fungal multidrug resistance ATP-binding cassette (ABC) transporters were identified based on the complete genome sequence and phylogenetic analysis of A. lentulus, which belonged to the ALDp subfamily, the PDR subfamily (AtrB, CDR1, and CDR2), and the MDR subfamily (MDR1), respectively, and there were four genes that are annotated to the major facilitator superfamily multidrug transporter. Further phylogenetic tree classification of the ABC transporter subfamilies predicted in the nine selected A. fumigatus complex strains showed that these putative ABC proteins were divided into two main clusters, which belonged to the PDR (CDR1, CDR2, AtrB, and AtrF) and MDR subfamilies (MDR1, MDR2, and MDR3). The distribution of these ABC proteins varies among different species of the A. fumigatus complex. Conclusions: The main result obtained from this study for the whole genome of A. lentulus provide new insights into better understanding the biological characteristics, pathogenicity, and resistance mechanisms of this bacterium. In this study, two resistance mechanisms, which include CYP51A gene mutation and multidrug-resistant ABC transporter, were predicted in a single isolate. Based on the predicted CYP51A-F46Y/N248T site mutation combination, we speculate that the CYP51A gene of A. lentulus may be partially responsible for azole resistance. Based on the predicted ABC transporter family genes, we hypothesize that resistance to multiple azoles in A. lentulus is mediated, at least in part, by these ABC transporters with resistance.

Rare case of aspergillosis with solitary renal involvement: micro-fungus ball in graft kidney.

Kidney transplant recipients are at an increased risk of various infections due to immunosuppressive medications. Among them, fungal infections are associated with high mortality and morbidity. This report presents the case of a 54-year-old kidney-transplant recipient who was diagnosed with aspergillosis with solitary renal involvement. He was diagnosed by kidney biopsy with the micro-fungus ball. In the biopsy sample, consisting mostly of the medulla, a small focus consisting of an aggregate of fungal microorganisms was identified. The micro-fungus ball, which was also present in serial sections, was characterized by slight pigmentation and septate hyphae with acute angle branching, highlighted by the silver stains. The patient was examined for invasive fungal infection. In CT scans, there were no signs of invasive fungal infection. Due to the unexpected kidney biopsy finding, the patient underwent a repeat allograft biopsy from which a culture was sent. Aspergillus fumigatus complex was detected in tissue fungal culture of this repeat biopsy. The patient was started on voriconazole treatment and was successfully treated. It should be kept in mind that fungal infections with isolated subtle renal involvement may be possible in KTR under immunosuppressive treatment without an obvious fungal focus being demonstrated by imaging methods.

Multilocus Sequence Analysis of Aspergillus Species from Clinical Samples and the Surge of Cryptic Aspergillosis in Southern India: A Descriptive Cross-sectional Study

Introduction: Aspergillus species cause a broad range of infections, from Allergic Bronchopulmonary Aspergillosis (ABPA) to invasive aspergillosis. Accurate species-level identification is crucial for diagnosis and treatment, especially since some rare Aspergillus species exhibit increased resistance to common antifungal agents. Aim: To identify Aspergillus species at the species complex level and determine the emergence of cryptic Aspergillus species using Multilocus Sequence Analysis (MLSA). Materials and Methods: This descriptive cross-sectional study was conducted over eight months, from January 2023 to August 2023, in three tertiary care centres in Chennai, India. Sixty Aspergillus strains were isolated from various clinical samples. The primary inclusion criteria were patients with suspected aspergillosis, categorised based on the European Organszation for Research and Treatment of Cancer Mycoses Study Group (EORTC-MSG) criteria. Isolates were grown on Oatmeal Agar or Sabouraud Dextrose Agar (SDA) and phenotypically identified using tease mount and slide culture techniques. Genomic fungal Deoxyribonucleic acid (DNA) was extracted and amplified using specific primers for Internal transcribed spacer (ITS), beta-tubulin, hydrophobin, and calmodulin genes, followed by nucleic acid sequencing. Results: Among the 60 Aspergillus isolates, the Aspergillus flavus complex comprised 26 (43.3%), the Aspergillus fumigatus complex 16 (26.7%), the Aspergillus niger complex 12 (20%), and the Aspergillus terreus complex 6 (10%). Initial speciation relied on microscopic and cultural characteristics. Isolates were recovered from various clinical specimens: bronchial wash 6 (10%), Bronchoalveolar lavage (BAL) fluid 10 (16.7%), sputum 3 (5%), ear swab 20 (33.3%), endotracheal aspirate 8 (13.3%), pus and tissue from the nasal cavity and paranasal sinus 11 (18.3%), and tissue from other sites 2 (3.3%). MLSA identified common species such as A. flavus, A. fumigatus, A. terreus, and A. niger, as well as rare species including A. awamori, A. carneus, A. lentulus, A. oryzae, A. tamarii, A. tubingensis, and A. welwitschiae. Sequence identity ranged from 99% to 100%, confirming species diversity. Phylogenetic analysis using ITS, beta-tubulin, hydrophobin, and calmodulin genes supported species identification with robust bootstrap support (1000 replications). Conclusion: Cryptic Aspergillus species are prevalent in clinical samples from the South Indian population, with a notable presence from the section Nigri and the first-time isolation of Aspergillus carneus in clinical samples from India. Molecular identification is essential for determining the invasive potential and guiding appropriate antifungal therapy.

Fungi involved in rhinosinusitis in arid regions: insights from molecular identification and antifungal susceptibility.

Fungal rhinosinusitis (FRS) is a common problem worldwide, with an increasing burden in arid climate regions. Aspergillus species are the most common causative agents involved. In the present study, we investigated the prevalence, molecular characterization, and antifungal susceptibility of opportunists causing FRS in Sudan on the basis of strains collected over a period of 5 years. β-Tubulin and calmodulin sequencing were used for species identification, and antifungal susceptibility profiles were evaluated by the protocol of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Predominant species belonged to the Aspergillus flavus complex (n = 244), A. terreus complex (n = 16), A. fumigatus complex (n = 7), and other fungi (n = 17). Molecular identification of 94 strains of Aspergillus revealed the following species: A. flavus (n = 88), A. terreus (n = 1), A. citrinoterreus (n = 2), A. fumigatus (n = 1), A. caespitosus (n = 1), and A. sydowii (n = 1). Several A. flavus and an A. fumigatus isolates showed reduced susceptibility to azoles (minimum inhibitory concentrations above the clinical breakpoints or epidemiological cutoff values). Despite several mutations revealed in cyp51A of these isolates, none could be directly linked to azole resistance. Molecular identification of fungi causing FRS is useful to identify cryptic species and for epidemiologic studies. IMPORTANCE Fungal rhinosinusitis (FRS) is a significant clinical problem in arid regions. This study provides new insights into the prevalence, etiology, and antifungal susceptibility of FRS pathogens in Sudan, where the disease burden is high. Aspergillus species, particularly the A. flavus complex, were identified as the primary FRS pathogens in the region, with some evidence of antifungal resistance. The molecular identification of fungal species causing FRS is useful for detecting antifungal resistance, identifying cryptic species, and characterizing the epidemiology of the disease. The emergence of Azole resistance Aspergilli in Sudan highlights the need for continued surveillance and appropriate use of antifungal agents. These findings have important implications for clinical management, public health policy, and future research on FRS. Publishing this study in Microbiology Spectrum would enable other researchers and clinicians to build on these findings, ultimately improving the diagnosis, treatment, and prevention of FRS.

A Tale of Two Hospitals: Comparing CAPA Infections in Two ICUs During the Spanish Fourth Pandemic Wave.

COVID-19 associated pulmonary aspergillosis (CAPA) is a new clinical entity linked to SARS-CoV-2 infection that is causing a rise on the risk of complications and mortality, particularly in critical patients. We compared diagnostic and clinical features in two cohorts of patients with severe COVID-19 admitted in the intensive care units (ICU) of two different hospitals in Madrid, Spain, between February and June 2021. Clinical and microbiological relevant aspects for CAPA diagnosis were collected for further classification. CAPA was classified as colonization, possible, probable, proven, and tracheobronchial aspergillosis according to the ECMM/ISHAM consensus, with some modifications to consider tracheobronchial aspirate as sample comparable to non-bronchoscopic lavages (NBL). 56 patients admitted in HULP (Hospital Universitario La Paz) ICU and 61 patients admitted in HEEIZ (Hospital de Emergencias Isabel Zendal) ICU had clinical suspicion of invasive fungal disease in the context of COVID-19 infection. Cultures were positive for Aspergillus spp. in 32 patients. According to 2020 European Confederation of Medical Mycology and the International Society for Human and Animal Mycology (ECMM/ISHAM) consensus, 11 patients were diagnosed with possible CAPA and 10 patients with probable CAPA. Global incidence for CAPA was 6.3%. Global median days between ICU admission and diagnosis was 14day. Aspergillus fumigatus complex was the main isolated species. Antifungal therapy was used in 75% of patients with CAPA suspicion, with inter-hospital differences in the administered antifungals. Global overall mortality rate for CAPA patients was 66.6% (14/21). All-cause mortality in non-CAPA cohorts were of 26.3% in HULP group (34/129) and 56.8% (104/183) in HEEIZ group. There were no significant differences in incidence between the two hospitals, and differences in antifungal therapy did not correlate with differences in mortality, reflecting that both first-line azoles and Amphotericin B could be effective in treating CAPA infections, according to the current guideline indications.

351. Epidemiology and antifungal susceptibilities of clinically significant filamentous fungi from a tertiary hospital in Singapore from 2018 – 2021

Abstract Background Filamentous fungi infections are associated with significant morbidity. Although an increase in antifungal resistance has been reported globally, no data pertaining to Singapore is available. This study investigates the species distribution and antifungal susceptibility profiles of molds isolated from a tertiary hospital in Singapore. Methods A retrospective review of fungal cultures with antifungal susceptibility results reported in the laboratory information system from 2018 to 2021 was conducted. Unique isolates per patient were included. Molds were identified primarily by morphology. Molecular studies were used as an adjunct where required. Susceptibility testing was performed via broth microdilution (Sensititre YeastOne) and gradient diffusion (Liofilchem) methods. Results 125 isolates were analyzed (Table 1). The most common molds recovered were Aspergillus, Fusarium and Mucorales. Aspergillus and Mucorales frequently involved the respiratory tract and skin and soft tissue (SSTI) whereas Fusarium was isolated from SSTI and blood. Amphotericin B had moderate activity against all molds, with only 11 out of 88 isolates (12.5%) being non-wild type (Table 2). The echinocandins exhibited good activity against Aspergillus and other hyaline molds but not Fusarium and Mucorales. The triazoles were most useful against Aspergillus, with posaconazole demonstrating the lowest geometric mean of 0.067. Fusarium had high MICs when tested against the azoles. Posaconazole was the azole of choice for Mucorales. Six Aspergillus isolates (all A. fumigatus) were non-wild type when tested against voriconazole, with a MIC >1. Of these six isolates, three were also non-wild type for amphotericin B, with a MIC >2. In particular, one Aspergillus fumigatus complex isolate demonstrated resistance across all tested azoles and sequencing revealed Cyp51 mutations. Conclusion Aspergillus species is the most prevalent clinically significant mold in our hospital. Although triazoles and echinocandins exhibit good activity, 13.33% of Aspergillus isolates were non-wild type for amphotericin B. Given the detection of these non-wild type Aspergillus species, susceptibility testing may be indicated in seriously ill patients to aid clinicians in selecting antifungal therapy. Disclosures All Authors: No reported disclosures.

Validity of Platelia Aspergillus IgG and Aspergillus Precipitin Test To Distinguish Pulmonary Aspergillosis from Colonization.

When Aspergillus, an ubiquitous, saprophytic fungus, is detected in respiratory tract specimens collected from chronic respiratory disease patients, it is important to determine whether it is a true infection or colonization. We investigated the usefulness of the Bio-Rad Platelia Aspergillus IgG (Platelia Aspergillus IgG) enzyme-linked immunosorbent assay (ELISA) method and the Aspergillus precipitin test to distinguish pulmonary aspergillosis from colonization. Between January 2017 and November 2021, 51 confirmed, untreated pulmonary aspergillosis (33 chronic pulmonary aspergillosis [CPA] and 18 allergic bronchopulmonary aspergillosis [ABPA]) and 77 colonization patients were included in this study. At first, the conventional cutoff value was utilized in assessing the validity of the two antibody tests for distinguishing pulmonary aspergillosis from colonization. The Platelia Aspergillus IgG cutoff value was then reevaluated to fit this situation. Finally, differences in test accuracy dependent on Aspergillus species were assessed for both antibody tests by comparing cases with Aspergillus fumigatus complex and those with non-fumigatus Aspergillus complex. Both antibody tests demonstrated significantly higher positive rates for pulmonary aspergillosis (P < 0.0001) than colonization. The cutoff value should be 15.7 arbitrary units (AU)/mL to best distinguish infection from colonization, which was higher than the conventional value of 10 AU/mL. The diagnostic sensitivity of Platelia Aspergillus IgG for the non-fumigatus Aspergillus complex was inferior to the A. fumigatus complex (P = 0.019). In conclusion, both Aspergillus antibody tests were valid to distinguish infection from colonization, although we should note the higher cutoff value for Platelia Aspergillus IgG and the lower sensitivity in cases of non-fumigatus Aspergillus infection. IMPORTANCE Pulmonary aspergillosis is the most common pulmonary fungal infection. However, Aspergillus is a ubiquitous, saprophytic fungus; it can be detected in respiratory specimens even in the absence of infection. Especially since Aspergillus is detected in respiratory specimens collected from patients with chronic respiratory disease, it is important to determine whether it is true infection or colonization. We investigated the validity of the Platelia Aspergillus IgG ELISA method and the Aspergillus precipitin test to distinguish pulmonary aspergillosis from colonization. Both antibody tests were considered useful in differentiating true infection from colonization in respiratory practice. The appropriate cutoff value for Platelia Aspergillus IgG was higher than the conventional value, and it was also noted that the sensitivity of both antibody tests for non-fumigatus Aspergillus complex was low. This study will be significant in real-world clinical practice of pulmonary aspergillosis using antibody tests in respiratory care.

Indoor environment assessment of special wards of educational hospitals for the detection of fungal contamination sources: A multi-center study (2019-2021).

The hospital environment was reported as a real habitat for different microorganisms, especially mold fungi. On the other hand, these opportunistic fungi were considered hospital-acquired mold infections in patients with weak immune status. Therefore, this multi-center study aimed to evaluate 23 hospitals in 18 provinces of Iran for fungal contamination sources. In total, 43 opened Petri plates and 213 surface samples were collected throughout different wards of 23 hospitals. All collected samples were inoculated into Sabouraud Dextrose Agar containing Chloramphenicol (SC), and the plates were then incubated at 27-30ºC for 7-14 days. A total of 210 fungal colonies from equipment (162, 77.1%) and air (48, 22.9%) were identified. The most predominant isolated genus was Aspergillus (47.5%), followed by Rhizopus (14.2%), Mucor (11.7%), and Cladosporium (9.2%). Aspergillus (39.5%), Cladosporium (16.6%), as well as Penicillium and Sterile hyphae (10.4% each), were the most isolates from the air samples. Moreover, intensive care units (38.5%) and operating rooms (21.9%) had the highest number of isolated fungal colonies. Out of 256 collected samples from equipment and air, 163 (63.7%) were positive for fungal growth. The rate of fungal contamination in instrument and air samples was 128/213 (60.1%) and 35/43 (81.2%), respectively. Among the isolated species of Aspergillus, A. flavus complex (38/96, 39.6%), A. niger complex (31/96, 32.3%), and A. fumigatus complex (15/96, 15.6%) were the commonest species. According to our findings, in addition to air, equipment and instrument should be considered among the significant sources of fungal contamination in the indoor environment of hospitals.

In Vitro Susceptibilities of Worldwide Isolates of Intrapulmonary Aspergillus Species and Important Candida Species in Sterile Body Sites against Important Antifungals: Data from the Antimicrobial Testing Leadership and Surveillance Program, 2017-2020.

To understand the changes of resistance in clinically commonly encountered fungi, we used the Antimicrobial Testing Leadership and Surveillance (ATLAS) database to explore in vitro antifungal susceptibilities against clinically important isolates of Aspergillus and Candida species (collected from intrapulmonary and sterile body areas, respectively). We applied the CLSI antifungal 2020 and the EUCAST antifungal 2020 guidelines. From 2017 to 2020, isolates of intrapulmonary Aspergillus fumigatus (n = 660), Aspergillus niger (n = 107), Aspergillus flavus (n = 96), Aspergillus terreus (n = 40), and Aspergillus nidulans species complex (n = 26) and sterile site-originated isolates of Candida albicans (n = 1,810), Candida glabrata (n = 894), Candida krusei (n = 120), Candida dubliniensis (n = 107), Candida lusitaniae (n = 82), Candida guilliermondii (n = 28), and Candida auris (n = 7) were enrolled in this study. Using the EUCAST 2020 breakpoints, it was demonstrated that amphotericin B and posaconazole displayed poor in vitro susceptibility rates against A. fumigatus isolates (<50% and 18.9%, respectively). In contrast, isavuconazole and itraconazole showed high in vitro potency against most Aspergillus isolates (>92%). Most intrapulmonary Aspergillus isolates exhibited MICs of ≤0.06 μg/mL to anidulafungin. Furthermore, intrapulmonary A. fumigatus isolates collected from Italy and the United Kingdom exhibited lower in vitro susceptibility to isavuconazole (72.2% and 69%, respectively) than those in the remaining ATLAS participant countries (>85%). Higher isavuconazole MIC90s against C. auris and C. guilliermondii (1 and 4 μg/mL, respectively) were observed compared to the other five Candida species. Despite the aforementioned MICs and susceptibilities against fungi, research needs to consider the pharmacokinetic (PK) profiles, pharmacodynamic (PD) parameters, and clinical treatment experience with antifungals against specific Aspergillus species. IMPORTANCE In addition to monitoring the antifungal susceptibilities of clinically important fungi, reviewing the PK/PD indices and the clinical therapy experience of antifungals under evaluation are important to guide an appropriate antifungal prescription. The efficacies of liposomal amphotericin B complex and anidulafungin for the treatment of pulmonary aspergillosis caused by different Aspergillus species need to be periodically evaluated in the future.

Morphologic identification of clinically encountered moulds using a residual neural network.

The use of morphology to diagnose invasive mould infections in China still faces substantial challenges, which often leads to delayed diagnosis or misdiagnosis. We developed a model called XMVision Fungus AI to identify mould infections by training, testing, and evaluating a ResNet-50 model. Our research achieved the rapid identification of nine common clinical moulds: Aspergillus fumigatus complex, Aspergillus flavus complex, Aspergillus niger complex, Aspergillus terreus complex, Aspergillus nidulans, Aspergillus sydowii/Aspergillus versicolor, Syncephalastrum racemosum, Fusarium spp., and Penicillium spp. In our study, the adaptive image contrast enhancement enabling XMVision Fungus AI as a promising module by effectively improve the identification performance. The overall identification accuracy of XMVision Fungus AI was up to 93.00% (279/300), which was higher than that of human readers. XMVision Fungus AI shows intrinsic advantages in the identification of clinical moulds and can be applied to improve human identification efficiency through training. Moreover, it has great potential for clinical application because of its convenient operation and lower cost. This system will be suitable for primary hospitals in China and developing countries.

Mixed invasive fungal infections among COVID-19 patients.

Background and Purpose: The healthcare system in India collapsed during the second wave of the COVID-19 pandemic. A fungal epidemic was announced amid the pandemic with several cases of COVID-associated mucormycosis and pulmonary aspergillosis being reported. However, there is limited data regarding mixed fungal infections in COVID-19 patients. Therefore, we present a series of ten consecutive COVID-19 patients with mixed invasive fungal infections (MIFIs).Materials and Methods: Among COVID-19 patients hospitalized in May 2021 at a tertiary care center in North India, 10 cases of microbiologically confirmed COVID-19-associated mucormycosis-aspergillosis (CAMA) were evaluated.Results: All patients had diabetes and the majority of them were infected with severe COVID-19 pneumonia (6/10, 60%) either on admission or in the past month while two were each of moderate (20%) and mild (20%) categories of COVID-19; and were treated with steroid and cocktail therapy. The patients were managed with amphotericin-B along with surgical intervention. In total, 70% of all CAMA patients (Rhizopus arrhizus with Aspergillus flavus in seven and Aspergillus fumigatus complex in three patients) survived.Conclusion: The study findings reflected the critical importance of a high index of clinical suspicion and accurate microbiological diagnosis in managing invasive dual molds and better understanding of the risk and progression of MIFIs among COVID-19 patients. Careful scrutiny and identification of MIFIs play a key role in the implementation of effective management strategies.

Consensus guidelines for the diagnosis and management of invasive aspergillosis, 2021.

Invasive aspergillosis (IA) in haematology/oncology patients presents as primary infection or breakthrough infection, which can become refractory to antifungal treatment and has a high associated mortality. Other emerging patient risk groups include patients in the intensive care setting with severe respiratory viral infections, including COVID-19. These guidelines present key diagnostic and treatment recommendations in light of advances in knowledge since the previous guidelines in 2014. Culture and histological-based methods remain central to the diagnosis of IA. There is increasing evidence for the utility of non-culture methods employing fungal biomarkers in pre-emptive screening for infection, as well as for IA diagnosis when used in combination. Although azole resistance appears to be uncommon in Australia, susceptibility testing of clinical Aspergillus fumigatus complex isolates is recommended. Voriconazole remains the preferred first-line antifungal agent for treating primary IA, including for extrapulmonary disease. Recommendations for paediatric treatment broadly follow those for adults. For breakthrough and refractory IA, a change in class of antifungal agent is strongly recommended, and agents under clinical trial may need to be considered. Newer immunological-based imaging modalities warrant further study, while surveillance for IA and antifungal resistance remain essential to informing the relevance of current treatment recommendations.

Stable prevalence of triazole-resistance in Aspergillus fumigatus complex clinical isolates in a Belgian tertiary care center from 2016 to 2020

BackgroundPrevalence reports of triazole-resistance in Aspergillus fumigatus differ between countries and centers and may likewise vary over time. Continuous local surveillance programs to establish the evolving epidemiology of triazole-resistance in A. fumigatus are crucial to guide therapeutic recommendations. Here, we determined the prevalence of triazole-resistance in A. fumigatus complex culture-positive patients at the tertiary care center University Hospitals Leuven in Belgium in clinical isolates from 2016 to 2020. MethodsAll A. fumigatus complex isolates cultured from UZ Leuven patients between 2016 and 2020 were screened for triazole-resistance. Confirmation of resistance to voriconazole, posaconazole and itraconazole was performed with the European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. Mutations in the cyp51A gene in triazole-resistant isolates were determined by sequencing. Patients were classified as susceptible or resistant cases based on their isolate's susceptibility phenotype. ResultsWe screened 2494 A. fumigatus complex isolates from 1600 patients (320 ± 38 [SD] patients per year). The prevalence of triazole-resistance in patients was 8.3% (28/337), 6.7% (26/386), 7.0% (21/301), 7.1% (21/294) and 7.4% (21/282) in 2016, 2017, 2018, 2019 and 2020 respectively, with an overall triazole-resistance prevalence of 7.1% (85/1192; 95% CI 6.6–7.7%). The TR34/L98H mutation was the most prevalent (83.0%, 78/94) with most isolates displaying resistance to all triazole antifungals tested (94.8%, 74/78). ConclusionThe prevalence of triazole-resistance in A. fumigatus has remained stable from 2016 to 2020 in our center ranging between 6.7 and 8.3%, with an overall five-year prevalence of 7.1%. The environmentally associated cyp51A gene mutations were most prevalent amongst triazole-resistant isolates and conferred resistance to all antifungals tested in 73% of the isolates.

Recovery and Its Dynamics of Filamentous Fungi from Clinical Specimen Cultures: An Extensive Study.

ABSTRACTThe culture method remains vital in diagnosing fungal infections, but extensive data-based evaluation of the method, especially for filamentous fungi (molds), is minimal. The purpose of this study was to characterize mold recoveries from fungal cultures and the impact of media and incubation duration. Clinical specimens for fungal cultures were submitted primarily from the eastern and central United States, and mold isolation data were prospectively collected and analyzed. A total of 1,821 molds in 59 genera were isolated from 1,687 positive specimens, accounting for approximately 5.6% of our cohort of 30,000 fungal cultures. Within 2 weeks, nearly 90% of molds and 97.3% of Aspergillus fumigatus complex were recovered (>95% confidence interval [CI]). All Mucorales fungi were recovered within 11 days of incubation. The recovery peak time was day 3 for Mucorales fungi, day 4 for hyaline molds, day 5 for dematiaceous molds, and day 7 for Onygenales fungi. The recovery of Histoplasma capsulatum and Trichophyton species in the fourth week of incubation reveals that a 3-week incubation time is insufficient. Inhibitory mold agar was the best medium for recovering all mold types among all tested specimen types, yielding nearly 78% of mold growth overall, indicating the necessity of selective medium for fungal cultures.IMPORTANCE Fungal culture is the gold standard method of diagnosing fungal infections, but important information, such as the impact of media and incubation times on fungal recovery, is not well documented. This study addressed these gaps using extensive data-based evaluation focused on molds. We identified the best medium types and incubation times for better fungal culture practice. We analyzed 1,821 molds from 1,687 positive specimens in our cohort of approximately 30,000 fungal cultures. Mold recovery peaked between 3 and 7 days of incubation, dependent upon the type of mold. Some well-defined fungal pathogens, such as Histoplasma capsulatum and Trichophyton species, were isolated in the fourth week of incubation. Inhibitory mold agar was identified as the best medium for recovering all mold types among all tested specimen sources. As we are aware, this is the largest study of fungal culture methods and supports 4 weeks of incubation for optimal mold recovery.

Detection of azole resistance in Aspergillus fumigatus complex isolates using MALDI-TOF mass spectrometry

ObjectivesThe main goal of this study was to accurately detect azole resistance in species of the Aspergillus fumigatus complex by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MethodsIdentification of isolates (n = 868) was done with MALDI-TOF MS using both commercial and in-house libraries. To determine azole susceptibility, the EUCAST E.Def. 9.3.2 method was applied as the reference standard. Identification of resistant isolates was confirmed by DNA sequence analysis. Protein spectra obtained by MALDI-TOF MS were analysed to differentiate species within the A. fumigatus complex and to detect azole-resistant A. fumigatus sensu stricto isolates. ResultsCorrect discrimination of A. fumigatus sensu stricto from cryptic species was accomplished in 100% of the cases applying principal component analysis (PCA) to protein spectra generated by MALDI-TOF MS. Furthermore, a specific peak (4586 m/z) was found to be present only in cryptic species. The application of partial least squares (PLS) discriminant analysis allowed 98.43% (±0.038) discrimination between susceptible and azole-resistant A. fumigatus sensu stricto isolates. Finally, based on PLS and SVM, A. fumigatus sensu stricto isolates with different cyp51A gene mutations were correctly clustered in 91.5% of the cases. ConclusionsMALDI-TOF MS combined with peak analysis is a novel tool that allows the differentiation of A. fumigatus sensu stricto from other species within the A. fumigatus complex, as well as the detection of azole-resistant A. fumigatus sensu stricto. Although further studies are still needed, the results reported here show the great potential of MALDI-TOF and machine learning for the rapid detection of azole-resistant Aspergillus fumigatus isolates from clinical origins.

Changing Trends in Isolation Frequencies and Species of Clinical Fungal Strains: What Do the 12-years (2008-2019) Mycology Laboratory Data Tell About?

The frequency and variety of infections caused by fungi are increasing. However, changes and intercenter and regional differences are observed in the distribution of fungal species over the years. It is important to update the epidemiological data in order to enable early and appropriate treatment. In this retrospective study, the number of fungi isolated from clinical samples, their distribution at the genus/ species level and the variations over the years in Hacettepe University hospital which is a regional center for patients at risk of fungal infection were investigated. For this purpose, laboratory records from 2008- 2019 were examined and 21813 fungal strains isolated from 19636 clinical samples were detected. When the first (2008-2013) and second (2014-2019) six-year periods were compared, a 2.5 fold increase was observed in the number of specimens yielding fungal growth (first period; n= 5620, second period; n= 14016). Fungi were most frequently isolated from urine (45.0%), lower respiratory tract (30.7%) and blood (6.8%) samples. Mould isolation rate in all samples increased significantly in the second six-year period (from 8.3% to 10.6%, p≤ 0.001). As expected, the most frequent yeast was Candida albicans (57.0%) and mould was Aspergillus fumigatus complex (50.4%). In the second six-year period, isolation of C.albicans (59.3% to 56.0%, p≤ 0.001) among yeasts and A.fumigatus complex (58.1% to 48.0%, p≤ 0.001) among moulds decreased significantly. In urine specimens, most common fungi were C.albicans (49.8%), Candida glabrata complex (15.6%), Candida tropicalis (8.9%) and Candida kefyr (7.5%). In lower respiratory tract specimens, the most common mould was A.fumigatus complex (51.2%), which has decreased from 63.7% in the first six years to 47.1% in the second period (p≤ 0.001). Over the same period, other Aspergillus species (from 25.5% to 34.1%, p= 0.002) and non-Aspergillus moulds (from 36.3% to 52.9%, p≤ 0.001) were increased. In blood samples, C.albicans (44.4%), Candida parapsilosis complex (21.5%) and C.glabrata complex (13.0%) were the most frequent species. In the second six-year period, the frequency of C.albicans decreased from 47.3% to 42.2% (p= 0.059) and the frequency of C.glabrata complex increased from 9.5% to 15.5% (p≤ 0.001) when compared to the first period. For the sterile specimens other than blood, the most common species were C.albicans (37.8%), C.glabrata complex (9.1%) and C.parapsilosis complex (4.7%). However, the number of fungal isolates and the distribution of the species showed great variation over the years. In our center, a substantial increase in the number of fungal strains isolated from the clinical specimens were observed over a 12-years period. In addition and similar to previously published reports, the increase of strains belonging to species with decreased antifungal susceptibility and/or species with unknown susceptibility were detected. The use of local data is required in order to implement early and appropriate antifungal treatment because of inter-center and regional differences observed in epidemiological trends regarding the distributions of fungal genera and species. Surveillance studies to be conducted with the participation of large and sufficient numbers of centers in our country, as we have done for our center, will also contribute to approaches regarding the management of fungal infections by revealing the epidemiological data in a comprehensive manner.

Dementia-Like Symptoms Associated With Posaconazole

Posaconazole is widely used in lung transplant recipients as pre-emptive therapy or universal fungal prophylaxis. In this patient group, posaconazole is increasingly used instead of voriconazole due to the concerns of an increased risk of squamous cell carcinoma (SCC) with voriconazole, particularly with its long-term use. Dose dependent toxicity has not been identified for posaconazole in the registration trials of intravenous (IV) and modified-release tablet formulations. This is supported by post-marketing experience. We describe a lung transplant recipient who experienced dementia-like symptoms almost 3 years after commencing posaconazole for treatment of Aspergillus fumigatus complex and Lomentospora prolificans (formerly Scedosporium prolificans) fungal infections. Symptoms resolved upon discontinuation of posaconazole, but recurred when re-challenged at a lower dose more than a year later. To the best of our knowledge, this is the first case reporting a dementia-like state with posaconazole.

Clinical and Microbiological Characterization of Invasive Pulmonary Aspergillosis Caused by Aspergillus lentulus in China.

Invasive aspergillosis (IA) due to Aspergillus lentulus is associated with high mortality. In this study, we investigated the clinical and microbiological characteristics of 6 fatal cases of proven or probable IA caused by A. lentulus in China. Underlying immunosuppression, prior antifungal exposure, and intensive care unit (ICU) hospitalization were important risk factors for invasive A. lentulus infection. Phenotypic differences were observed for A. lentulus isolates including slower growth, reduced sporulation, and inability to grow at 48°C, compared with Aspergillus fumigatus complex. ITS sequencing was unable to distinguish A. lentulus from A. fumigatus, but sequencing of the benA, CaM, and rod A loci enabled reliable distinction of these closely related species. Phylogenetic analysis further confirmed that the ITS region had little variation within the Aspergillus section Fumigati while the benA gene offered the highest intraspecific discrimination. Microsatellite typing results revealed that only loci on chromosomes 1, 3, 5, and 6b generated detectable amplicons for identification. All A. lentulus isolates showed in vitro resistance to multiple antifungal drugs including amphotericin B (MIC range 4 to 8 μg/ml), itraconazole (MIC 2 μg/ml), voriconazole (MIC of 4–16 μg/ml), and posaconazole (MIC of 0.5–1 μg/ml). However, MECs for the echinocandin drugs ranged from 0.03–0.25, ≤0.008–0.015, and ≤0.015–0.03 μg/ml for caspofungin, micafungin, and anidulafungin, respectively. A. lentulus is an emerging fungal pathogen in China, causing fatal disease, and clinicians as well as laboratories should be alert to their increasing presence.

Molecular Identification of Aspergillus Fumigatus Complex from Lung Transplant Recipients Using Multi Locus Sequencing Analysis (MLSA)

Aspergillus fumigatus is responsible for Invasive aspergillosis (IA). The role of other closely related species such as Aspergillus lentulus and Aspergillus viridinutans which are members of the section Fumigati (A. fumigatus complex) in the setting of pre-emptive therapy in lung transplant recipients is not well defined, and may be associated with poor clinical response. A total of 99 A. fumigatus isolates that were identified by the mycology laboratory at Mount Sinai Hospital (MSH) using conventional phenotypic procedures were genotyped using Multilocus Sequencing Analysis (MLSA) of the β-tubulin (benA), hydrophobin (rodA), and calmodulin (cmd) gene. These isolates were originally isolated from BAL or sputum specimens collected from immunocompromised patients. Of the 99 isolates, 71, 12, and 16 were isolated from post-lung transplant recipients (LRT), pre-transplant recipients, and non-lung transplants, respectively. 98 isolates (99.8%) carried the DNA sequences of the aforementioned genes, which were identical to those of A. fumigatus reference strains in the GeneBank, and thus clustered in the same clade with A. fumigatus. Only one variant isolate from a case of disseminated infection was identified and clustered with the same clade as the fumigati reference species; A. thermomutatus. Four reference strains were processed along with our isolates in this study to ensure the sensitivity and specificity of the assays. The MLSA data analysis revealed that A. fumigatus were the only strains recovered in our population of lung transplant recipients. The MLSA results correlated very well with those obtained by conventional mycological procedures. A. fumigatus appears to be the main causative agent of Invasive aspergillosis in our LTRs in the setting of pre-emptive therapy. Other fumigati species were not detected in LTRs.