Reverse-transcriptase real-time PCR in the diagnostic strategy for invasive infections caused by Aspergillus fumigatus.

The aim was to develop an RT-qPCR targeting Aspergillus fumigatus and compare its performance to that of Aspergillus fumigatus qPCR for the diagnosis of invasive aspergillosis (IA). Samples from patients of the Lyon University hospitals for whom a suspicion of IA led to the realization of an Aspergillus fumigatus qPCR molecular diagnostic test over a 2-year period were included. The patients were classified according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC-MSGERC) criteria for suspected IA; RT-qPCR and qPCR assays were performed on all included samples. The sensitivities and specificities of RT-qPCR and qPCR were calculated and compared using the results of the EORTC-MSGERC classification as reference. The cycle threshold (Ct) results were compared according to IA classification and sample type. Among the 193 samples analyzed, 91 were classified as IA excluded, 46 as possible IA, 53 as probable IA, and 3 as proven IA. For all sample types, RT-qPCR was significantly more sensitive than qPCR for all IA classifications with an additional 17/102 samples detected (P-value < 0.01). For plasma samples, sensitivity was significantly higher and specificity significantly lower using RT-qPCR for all IA classifications (P-value < 0.001). The mean Ct obtained with RT-qPCR were significantly lower than those obtained with qPCR for all IA classifications and all sample types (P-value < 0.001 and P-value < 0.0001, respectively). RT-qPCR presents a higher sensitivity than qPCR for the diagnosis of IA due to Aspergillus fumigatus, particularly in samples with an intrinsically low fungal load.IMPORTANCEAspergillus fumigatus belongs to the critical priority group of the World Health Organization fungal priority pathogens list. Invasive aspergillosis (IA) is a life-threatening infection with poor prognosis and challenging diagnosis. PCR has been integrated into the 2020 European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus definitions for IA diagnosis. However, due to frequent low fungal burdens, its sensitivity needs to be improved. This work presents an innovative method for detecting total nucleic acids, corresponding to both ribosomal RNA and DNA, that enables IA diagnosis with greater sensitivity than conventional techniques, especially in non-invasive samples such as blood, enhancing the monitoring of this infection in high-risk patients.

Fungal Integrated Histomolecular Diagnosis Using Targeted Next-Generation Sequencing on Formalin-Fixed Paraffin-Embedded Tissues.

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Outcome of patients with cystic fibrosis colonized by Scedosporium and Lomentospora species: A longitudinal cohort study.

Scedosporium and Lomentospora species rank second among the filamentous fungi colonizing the airways of cystic fibrosis (CF) patients. These fungi could be responsible for allergic bronchopulmonary mycosis (ABPM) and bronchitis before lung transplantation and invasive infections after. However, their role in CF lung disease is debated. This study aimed to identify clinical or environmental factors associated with an airway colonization by Scedosporium/Lomentospora species in patients with CF over a period of 7 years. A longitudinal cohort study was conducted from 2008 to 2014 in the CF reference centre in Lyon, France, to compare the characteristics of patients with Scedosporium/Lomentospora colonized and non-colonized patients. During the study period, 283 patients completed the clinical and microbiological follow-up. The analysis revealed that a higher number and duration of hospitalizations, an increased number of courses of parenteral antibiotic therapy, a history of ABPA, and treatment by itraconazole were significantly associated with an airway colonization by Scedosporium/Lomentospora species. The rate of decline of forced expiratory volume in the first second was not statistically different between colonized and non-colonized patients. This study provides evidence that patients colonized by Scedosporium/Lomentospora species require more medical care than non-colonized patients. Additional care could be in part explained by the management of Scedosporium/Lomentospora-related diseases such as ABPM or bronchitis. However, we did not demonstrate a faster rate of decline of respiratory function or body mass index in colonized patients, suggesting, as previously reported, that colonization of the airways by these fungi does not play a significant role in the progression of CF disease.

This prospective study did not demonstrate a faster rate of decline of respiratory function or body mass index in cystic fibrosis (CF) patients colonized by Scedosporium/Lomentospora species compared to non-colonized patients, suggesting that these fungi do not play a significant role in the progression of CF disease.

Use of 1,3-Beta-D-Glucan concentration in peritoneal fluid for the diagnosis of intra-abdominal Candidiasis in Critically-ill patients.

Intra-Abdominal Candidiasis (IAC) is frequent and associated with high mortality in intensive care unit (ICU) patients. Antifungal treatments may be overused due to a lack of diagnostic tools to rule out IAC. Serum 1,3-Beta-D-Glucan (BDG) concentrations are used to diagnose Candida infections, its concentration in peritoneal fluid (PF) may help to confirm or invalidate the diagnosis of IAC. We performed a non-interventional, prospective, multicenter study, at the Hospices Civils de Lyon, France, in seven ICU located in three different hospitals from December 2017 to June 2018. IAC was defined as the isolation of Candida in a sample collected from the intra-abdominal cavity under sterile conditions in patients displaying clinical evidence of intra-abdominal infection. Among the 113 included patients, 135 PF samples corresponding to 135 intra-abdominal infection episodes were collected and BDG concentrations were assessed. IAC accounted for 28 (20.7%) of the intra-abdominal infections. Antifungals were administered empirically to 70 (61.9%) patients; among them, 23 (32.9%) had an IAC. The median [IQR] BDG value was significantly higher in IAC (8100 [3000;15000] pg/mL) than in non-IAC samples (1961 [332;10650] pg/mL). BDG concentrations were higher in PF with Fecaloid aspect and in case of positive bacterial culture. For a BDG threshold of 125 pg/mL, the negative predictive value to assess IAC was 100%. In conclusion, low BDG PF concentrations could be used to rule out IAC. https://clinicaltrials.gov/ct2/show/NCT03469401.

Intra-Abdominal Candidiasis (IAC) is associated with a high mortality in Intensive Care Unit (ICU) patients. 1,3-Beta-D-Glucan (BDG), a component of Candida cell wall, was prospectively measured in peritoneal fluid from ICU patients Low peritoneal BDG concentrations may be used to rule out IAC.

Invasive bone and joint infections from the French Scedosporiosis/lomentosporiosis Observational Study (SOS) cohort: no mortality with long-term antifungal treatment and surgery.

Little is known about localized osteoarticular Scedosporiosis (LOS). Most data come from case reports and small case series. Here we present an ancillary study of the nationwide French Scedosporiosis Observational Study (SOS), describing 15 consecutive cases of LOS diagnosed between January 2005 and March 2017. Adult patients diagnosed with LOS defined by osteoarticular involvement without distant foci reported in SOS were included. Fifteen LOS were analyzed. Seven patients had underlying disease. Fourteen patients had prior trauma as potential inoculation. Clinical presentation was arthritis (n = 8), osteitis (n = 5), and thoracic wall infection (n = 2). The most common clinical manifestation was pain (n = 9), followed by localized swelling (n = 7), cutaneous fistulization (n = 7), and fever (n = 5). The species involved were Scedosporium apiospermum (n = 8), S. boydii (n = 3), S. dehoogii (n = 1), and Lomentospora prolificans (n = 3). The species distribution was unremarkable except for S. boydii, which was associated with healthcare-related inoculations. Management was based on medical and surgical treatment for 13 patients. Fourteen patients received antifungal treatment for a median duration of 7 months. No patients died during follow-up. LOS exclusively occurred in the context of inoculation or systemic predisposing factors. It has a non-specific clinical presentation and is associated with an overall good clinical outcome, provided there is a prolonged course of antifungal therapy and adequate surgical management.

Localized osteoarticular scedosporiosis mostly occurs following direct inoculation. Management was most often based on voriconazole therapy and concomitant surgery. Unlike other invasive scedosporiosis, no patient died during follow-up.

Pulmonary aspergillosis in critically ill patients with Coronavirus Disease 2019 (COVID-19).

Occurrence of putative invasive pulmonary aspergillosis was screened in 153 consecutive adult intensive care unit (ICU) patients with respiratory samples addressed for mycological diagnosis during a 6-week period at the emergence of coronavirus disease 2019 (COVID-19) pandemic. Positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) polymerase chain reaction (PCR) was observed for 106 patients (69.3%). Nineteen of them (17.9%) with positive Aspergillus results were considered as having putative invasive pulmonary aspergillosis. These observations underline the risk of pulmonary aspergillosis in COVID-19 patients, even in patients not previously known to be immunosuppressed, advocating active search for Aspergillus infection and prompt antifungal treatment. Standardized surveillance protocols and updated definitions for ICU putative invasive pulmonary aspergillosis are needed. LAY ABSTRACT: Adult ICU patients with respiratory samples addressed for mycological diagnosis were screened during the emergence of COVID-19 pandemic. Positive SARS-CoV-2 PCR was observed for 106 patients, nineteen of them (17.9%) having aspergillosis. This underlines the risk of aspergillosis in COVID-19 patients.

K3-EDTA differentially inhibits the growth of Candida strains according to their azole resistance status.

The diagnosis of the life-threatening invasive Candida infections is mainly established using culture of specimens that might be collected on different devices including ethylene diamine tetraacetic acid (EDTA)-coated tubes. Despite the knowledge that EDTA inhibits bacterial cultures, and its use to treat oral fungal infections, its impact on Candida cultures has not been completely assessed. This study aimed at assessing it on azole-resistant and azole-susceptible strains. Clinical and American Type Culture Collection (ATCC) strains for Candida albicans (CA), C. glabrata (CGS), C. krusei (CK), azole-susceptible and azole-resistant strains of C. glabrata (CGS and CGR), C. lipolytica (CL), and C. inconspicua (CI) were characterized using MALDI-TOF MS and susceptibility testing and then incubated (1) with serial dilutions of tripotassic EDTA (0%-500% of the concentration in a sample tube) for 2 hours before plating onto ChromID Can2 agar; (2) for 0, 2, 4, 6, 7, or 8 hours at EDTA concentrations at 20% and 33% before seeding; and (3) with sodium citrate or lithium heparinate instead of EDTA for 2 hours before plating. After 48 hours at 35°C, colony-forming units were automatically quantified. An inhibitory effect of EDTA was observed, at different concentrations, for CA (20%), CGS (100%), and CGR (500%) (P < .05), but none was observed for CL, CI, and CK. The effect increased with incubation duration, at a faster rate for azole-susceptible strains. K3-EDTA inhibits Candida growth and EDTA-coated tubes should not be used for mycological culture-based analyses. The correlation between EDTA inhibition and Candida azole-resistance offers perspectives for the development of selective agar and new antifungal strategies.

Rare and unusual presentation of Cladophialophora infection in a pulmonary transplant cystic fibrosis patient.

A 35-year-old woman with severe cystic fibrosis was admitted for sudden loss of strength in both legs, revealing a myelitis. The medullary lesion biopsy revealed phaeohyphomycosis caused by Cladophialophora species. Myelitis caused by Cladophialophora bantiana is a rare disease associated with high mortality.

Acute graft-versus-host disease, invasive aspergillosis and Clostridium difficile colitis after peripheral blood stem cell transplantation: A complex network of causalities and a challenge for prevention.

Graft-versus-host disease (GVHD) is a known risk factor for invasive aspergillosis (IA), but remains poorly studied in relation to Clostridium difficile infection (CDI). We report a case of a 58-years-old patient who developed an IA within a protected room, CDI and GVHD after allogeneic allogeneic peripheral blood stem cell transplantation (PBSCT). Factors associated with this complex condition in patients receiving allogeneic PBSCT need to be identified.

Reappraising Cladophialophora bantiana phaeohyphomycosis in France: retrospective nation-based study.

BACKGROUND: Cladophialophora bantiana is one of the most virulent phaeohyphomycetes, typically causes non-angiogenic single (or sometimes multiple) cystic brain lesions, and has resulted in a mortality rate of up to 70%. Most C bantiana cases are described either in a series of isolated reports or in very small cohorts. The aim of this retrospective nation-based study was to share the data on C bantiana phaeohyphomycosis cases reported in France and French overseas territories over the past two decades to improve understanding of this disease. METHODS: Patients with C bantiana infection were processed through the active surveillance programme of invasive fungal infections launched by the National Reference Center for Mycoses and Antifungals, Institut Pasteur (Paris, France), and the French Surveillance Network of Invasive Fungal Infections, which involved 29 hospitals from mainland France and overseas French territories. Only proven and probable cases of infection, according to the revised and updated consensus definitions from the European Organization for Research and Treatment of Cancer and Mycoses Study Group, were included in the study. Patients were diagnosed or confirmed, or both, using a polyphasic approach at the Institut Pasteur between 2002 and 2022. Patients were separated into two groups: those with CNS involvement and those with no CNS involvement. The primary outcome was the survival rate. FINDINGS: A total of 23 patients with a C bantiana invasive infection were included during the study period (Jan 1, 2002, to Dec 31, 2022). The median age was 56 years in the CNS involvement group and 65 years in the non-CNS involvement group. Until 2021, the annual number of cases varied between zero and two, with six cases observed in 2022, the warmest year recorded in France since 1900. CNS involvement was observed in 15 (65%) patients, including three disseminated cases; skin and soft tissue involvement in seven (30%) patients and an isolated lung infection in one case. Diabetes was observed in five patients, and any immunodepression factor was observed in 14 (61%) of 23 patients. When considering only patients with CNS involvement, 9-month survival appeared higher in patients who underwent exeresis or large drainage (three [75%] of four patients vs three [27%] of 11 patients; p=0·24) and significantly higher in those treated for 2 or more weeks with triple antifungal therapy (liposomal amphotericin B plus posaconazole and flucytosine; seven [78%] of nine patients vs one [17%] of six patients; p=0·040). Two patients were treated with excision surgery alone (one patient with success, and the other patient lost to follow-up). INTERPRETATION: This study shows that the clinical presentations and underlying medical conditions of C bantiana infections are more diverse than previously described. It also emphasises a significant difference in mortality rate between those with and without CNS involvement. The prognosis improved when surgery was performed and triple antifungal therapy was administered. Such rare and devastating invasive fungal infections should be managed by a multidisciplinary team. FUNDING: Santé Publique France.

Features of cryptococcosis among 652 HIV-seronegative individuals in France: a cross-sectional observational study (2005-2020).

OBJECTIVES: We aimed to describe features and outcomes of cryptococcosis among HIV-seronegative individuals in a large surveillance network for cryptococcosis in France. METHODS: We included incident cases of cryptococcosis in HIV-seronegative individuals from 2005 to 2020. We compared patient characteristics, disease presentations, cryptococcal antigen results, and induction antifungal treatments according to underlying disease. We examined factors associated with 90-day mortality. Among patients with disseminated infections, we investigated whether receipt of flucytosine and polyene combination was associated with lower mortality. RESULTS: Among 652 individuals, 209 (32.1%) had malignancy, 130 (19.9%) were solid-organ transplant recipients, 204 (31.3%) had other immunocompromising conditions, and 109 (16.7%) had no reported underlying factor. The commonest presentations were disseminated infections (63.3%, 413/652) and isolated pulmonary infections (25.3%, 165/652). Solid-organ transplant patients were most likely to have disseminated infections and a positive serum cryptococcal antigen result. Patients with malignancy were older and less likely to receive a flucytosine-containing regimen for disseminated infections than others (58.7%, 78/133 vs. 73.2%, 194/265; p 0.029). The crude 90-day case-fatality ratio was 27.2% (95% CI, 23.5%-31.1%). Age ≥60 years (aOR: 2.75 [1.78-4.26]; p < 0.001), meningitis/fungaemia (aOR: 4.79 [1.80-12.7]; p 0.002), and malignancy (aOR: 2.4 [1.14-5.07]; p 0.02) were associated with higher 90-day mortality. Receipt of flucytosine and polyene combination was associated with lower 90-day mortality (aOR: 0.40 [0.23-0.71]; p 0.002) in multivariable analysis and inverse probability of treatment weighted analysis (aOR: 0.45 [0.25-0.80]; p 0.006). DISCUSSION: HIV-seronegative individuals with cryptococcosis comprise a wide range of underlying conditions with different presentations and outcomes, requiring a tailored approach to diagnosis and management.

Cryptococcus neoformans Infections Differ Among Human Immunodeficiency Virus (HIV)-Seropositive and HIV-Seronegative Individuals: Results From a Nationwide Surveillance Program in France.

Among 1107 cryptococcosis cases from the French surveillance network (2005-2020), the proportion of HIV-seronegative individuals has recently surpassed that of HIV-seropositive individuals. We observed marked differences in patient characteristics, disease presentations, cryptococcal antigen results, infecting species, and mortality according to HIV serostatus.

Characterization of Lung Inflammatory Response to Aspergillus fumigatus Spores.

The airway exposure to Aspergillus fumigatus spores (AFsp) is associated with an inflammatory response, potentially leading to allergic and/or chronic pulmonary aspergillosis. The aim of our study is to better understand the host response, first in vitro, then in vivo, following the chronic exposure of mice to AFsp. We investigated the inflammatory response to AFsp in cell mono- and co-culture systems with murine macrophages and alveolar epithelial cells. The mice were subjected to two intranasal instillations using 105 AFsp. Their lungs were processed for inflammatory and histopathological analyses. In cell culture, the gene expressions significantly increased for TNF-α, CXCL-1, CXCL-2, IL-1ß, IL-1α and GM-CSF in macrophages, with these increases being limited for TNF-α, CXCL-1 and IL-1α in epithelial cells. In co-culture, increases in the TNF-α, CXCL-2 and CXCL-1 gene expressions were observed to be associated with increased protein levels. The in vivo lung histological analyses of mice challenged by AFsp showed cellular infiltrates in the peribronchial and/or alveolar spaces. A Bio-Plex approach on the bronchoalveolar lavage revealed significant increases in the protein secretion of selected mediators of the challenged mice compared to the unchallenged mice. In conclusion, the exposure to AFsp resulted in a marked inflammatory response of macrophages and epithelial cells. These inflammatory findings were confirmed in mouse models associated with lung histologic changes.

Fatal Community-Acquired Peritonitis Due to Invasive Necrotizing Intestinal Mucormycosis: Case Report and Review of the Literature.

We report here a rare case of fatal rapidly progressive necrotizing gastrointestinal mucormycosis due to Mucor circinelloides f. circinelloides in the setting of community-acquired peritonitis, in an immunocompromised adult patient. Diagnosis was established by direct examination of peritoneal fluid showing hyphae consistent with mucormycosis confirmed by the culture.

Fungal infections in mechanically ventilated patients with COVID-19 during the first wave: the French multicentre MYCOVID study.

BACKGROUND: Patients with severe COVID-19 have emerged as a population at high risk of invasive fungal infections (IFIs). However, to our knowledge, the prevalence of IFIs has not yet been assessed in large populations of mechanically ventilated patients. We aimed to identify the prevalence, risk factors, and mortality associated with IFIs in mechanically ventilated patients with COVID-19 under intensive care. METHODS: We performed a national, multicentre, observational cohort study in 18 French intensive care units (ICUs). We retrospectively and prospectively enrolled adult patients (aged ≥18 years) with RT-PCR-confirmed SARS-CoV-2 infection and requiring mechanical ventilation for acute respiratory distress syndrome, with all demographic and clinical and biological follow-up data anonymised and collected from electronic case report forms. Patients were systematically screened for respiratory fungal microorganisms once or twice a week during the period of mechanical ventilation up to ICU discharge. The primary outcome was the prevalence of IFIs in all eligible participants with a minimum of three microbiological samples screened during ICU admission, with proven or probable (pr/pb) COVID-19-associated pulmonary aspergillosis (CAPA) classified according to the recent ECMM/ISHAM definitions. Secondary outcomes were risk factors of pr/pb CAPA, ICU mortality between the pr/pb CAPA and non-pr/pb CAPA groups, and associations of pr/pb CAPA and related variables with ICU mortality, identified by regression models. The MYCOVID study is registered with ClinicalTrials.gov, NCT04368221. FINDINGS: Between Feb 29 and July 9, 2020, we enrolled 565 mechanically ventilated patients with COVID-19. 509 patients with at least three screening samples were analysed (mean age 59·4 years [SD 12·5], 400 [79%] men). 128 (25%) patients had 138 episodes of pr/pb or possible IFIs. 76 (15%) patients fulfilled the criteria for pr/pb CAPA. According to multivariate analysis, age older than 62 years (odds ratio [OR] 2·34 [95% CI 1·39-3·92], p=0·0013), treatment with dexamethasone and anti-IL-6 (OR 2·71 [1·12-6·56], p=0·027), and long duration of mechanical ventilation (>14 days; OR 2·16 [1·14-4·09], p=0·019) were independently associated with pr/pb CAPA. 38 (7%) patients had one or more other pr/pb IFIs: 32 (6%) had candidaemia, six (1%) had invasive mucormycosis, and one (<1%) had invasive fusariosis. Multivariate analysis of associations with death, adjusted for candidaemia, for the 509 patients identified three significant factors: age older than 62 years (hazard ratio [HR] 1·71 [95% CI 1·26-2·32], p=0·0005), solid organ transplantation (HR 2·46 [1·53-3·95], p=0·0002), and pr/pb CAPA (HR 1·45 [95% CI 1·03-2·03], p=0·033). At time of ICU discharge, survival curves showed that overall ICU mortality was significantly higher in patients with pr/pb CAPA than in those without, at 61·8% (95% CI 50·0-72·8) versus 32·1% (27·7-36·7; p<0·0001). INTERPRETATION: This study shows the high prevalence of invasive pulmonary aspergillosis and candidaemia and high mortality associated with pr/pb CAPA in mechanically ventilated patients with COVID-19. These findings highlight the need for active surveillance of fungal pathogens in patients with severe COVID-19. FUNDING: Pfizer.

Azole resistance in Aspergillus fumigatus isolates from respiratory specimens in Lyon University Hospitals, France: prevalence and mechanisms involved.

Resistance of Aspergillus fumigatus to triazoles has been reported increasingly in Europe. As few data are available from Southern France, the objectives of this study were to assess the burden of A. fumigatus isolates with azole resistance from clinical specimens in Lyon, and explore the resistance mechanisms involved. In this retrospective cross-sectional study, 221 consecutive A. fumigatus isolates from respiratory samples were identified from an 8-month period from 195 patients attending the Pulmonary Medicine Departments of Lyon University Hospitals. Morphological identification was confirmed by sequence analysis of the ß-tubulin gene. All samples were tested for susceptibilities to itraconazole, voriconazole, posaconazole and isavuconazole using concentration gradient strips, and the results were confirmed using the EUCAST broth microdilution method. Resistance mechanisms were investigated by sequencing the cyp51A gene and its promoter, and by expression analysis of cyp51 and genes encoding several efflux transporters. Four isolates exhibited azole resistance. Three isolates presented with polymorphisms in an intronic region of cyp51A, and one isolate had F46Y, M172V and E427K polymorphisms. No mutations were identified in the cyp51A promoter, but significant induction of cyp51A and cyp51B gene expression was observed for all four and three isolates, respectively. Significant induction of atrF and cdr1B gene expression was observed for two and three isolates, respectively. No significant induction of MDR1/2/3/4, MFS56 and M85 gene expression was observed. To conclude, the observed prevalence of azole resistance was 2.1%. Significant induction of expression of the cyp51 genes and two genes encoding efflux transporters was evidenced, underlying the diversity of resistance mechanisms to be explored.

COVID-19-Associated Pulmonary Aspergillosis, Fungemia, and Pneumocystosis in the Intensive Care Unit: a Retrospective Multicenter Observational Cohort during the First French Pandemic Wave.

The aim of this study was to evaluate diagnostic means, host factors, delay of occurrence, and outcome of patients with COVID-19 pneumonia and fungal coinfections in the intensive care unit (ICU). From 1 February to 31 May 2020, we anonymously recorded COVID-19-associated pulmonary aspergillosis (CAPA), fungemia (CA-fungemia), and pneumocystosis (CA-PCP) from 36 centers, including results on fungal biomarkers in respiratory specimens and serum. We collected data from 154 episodes of CAPA, 81 of CA-fungemia, 17 of CA-PCP, and 5 of other mold infections from 244 patients (male/female [M/F] ratio = 3.5; mean age, 64.7 ± 10.8 years). CA-PCP occurred first after ICU admission (median, 1 day; interquartile range [IQR], 0 to 3 days), followed by CAPA (9 days; IQR, 5 to 13 days), and then CA-fungemia (16 days; IQR, 12 to 23 days) (P < 10-4). For CAPA, the presence of several mycological criteria was associated with death (P < 10-4). Serum galactomannan was rarely positive (<20%). The mortality rates were 76.7% (23/30) in patients with host factors for invasive fungal disease, 45.2% (14/31) in those with a preexisting pulmonary condition, and 36.6% (34/93) in the remaining patients (P = 0.001). Antimold treatment did not alter prognosis (P = 0.370). Candida albicans was responsible for 59.3% of CA-fungemias, with a global mortality of 45.7%. For CA-PCP, 58.8% of the episodes occurred in patients with known host factors of PCP, and the mortality rate was 29.5%. CAPA may be in part hospital acquired and could benefit from antifungal prescription at the first positive biomarker result. CA-fungemia appeared linked to ICU stay without COVID-19 specificity, while CA-PCP may not really be a concern in the ICU. Improved diagnostic strategy for fungal markers in ICU patients with COVID-19 should support these hypotheses. IMPORTANCE To diagnose fungal coinfections in patients with COVID-19 in the intensive care unit, it is necessary to implement the correct treatment and to prevent them if possible. For COVID-19-associated pulmonary aspergillosis (CAPA), respiratory specimens remain the best approach since serum biomarkers are rarely positive. Timing of occurrence suggests that CAPA could be hospital acquired. The associated mortality varies from 36.6% to 76.7% when no host factors or host factors of invasive fungal diseases are present, respectively. Fungemias occurred after 2 weeks in ICUs and are associated with a mortality rate of 45.7%. Candida albicans is the first yeast species recovered, with no specificity linked to COVID-19. Pneumocystosis was mainly found in patients with known immunodepression. The diagnosis occurred at the entry in ICUs and not afterwards, suggesting that if Pneumocystis jirovecii plays a role, it is upstream of the hospitalization in the ICU.

Aspergillus endophthalmitis: Potential role for vitreous galactomannan testing?

Eye damage during invasive aspergillosis is rarely described and biological diagnosis remains challenging. Here we report the case of a heart transplant recipient with ocular aspergillosis complicating disseminated aspergillosis. Although voriconazole was rapidly given, a decrease in visual acuity of the right eye was consistent with endophthalmitis, resulting in an emergency vitrectomy. The diagnosis was rapidly confirmed: laboratory results showed the presence of Aspergillus fumigatus in a vitreous sample. A series of systemic antifungal medications (liposomal amphotericin B, caspofungin, and voriconazole), several liposomal amphotericin B ocular injections, and pars plana vitrectomy resulted in a limited positive clinical outcome. Interestingly although standard mycological follow-up procedures were negative, Aspergillus antigen testing gave an index of 5.92 on vitreous humour, thus a new intraocular injection of liposomal amphotericin B was performed and voriconazole reinitiated. Ten other vitreous samples from patients without fungal infections were also tested, all showing indexes below 0.25. Although larger studies are needed, this case illustrates that galactomannan testing of vitreous humour could be useful for the diagnosis of fungal endophthalmitis if these data are confirmed in other patients, in particular, if standard mycology is negative and PCR is not available.

Fast-Growing Alveolar Echinococcosis Following Lung Transplantation.

Alveolar echinococcosis is a rare but life-threatening infection caused by the parasite Echinococcus multilocularis. Its natural history is characterized by a slow parasitic growth over several years. Increased incidence and shorter development delay have been reported in immune-compromised patients. We report the reactivation of aborted lesions within 12 months of lung transplantation leading to a fast-growing aggressive hepatic lesion. Timely identification of alveolar echninococcosis allowed prompt albendazole treatment and radical surgery leading to a favorable outcome 42 months after transplantation. However, close clinical, serological and radiological monitoring is required to rule out relapses in the long term. The pre-existence of aborted self-limited lesions of alveolar echinococcosis and the possibility for their atypical rapid growth in patients undergoing profound immunosuppression should be known by healthcare providers, even if working in non-endemic areas.

[Update on the diagnosis of parasitic and fungal infections]. / Actualités du diagnostic des infections parasitaires et fongiques.

The diagnosis of parasitic and fungal infections, historically based on the detection of these pathogens using direct diagnosis (macro/microscopic examination, culture) or serological methods, has considerably evolved in the last decades, especially with the development of molecular approaches and mass spectrometry. These techniques, as well as most analyses of parasitic and fungal serology, are mostly the preserve of Hospital University Centers Parasitology-Mycology laboratories. In 2016, the French association of medical parasitology and mycology teachers and hospital practitioners (Anofel) has provided a Catalogue of rare analyses, regularly updated and freely accessible on the Anofel website (https://anofel.net/). This tool, which hinges on 4 parts (parasitology, parasitic serology, mycology, and fungal serology), aims to provide information on all available analyses, and a list of hospital laboratories able to undertake them. It is complementary to the other reference works that were developed by our association, including the Guide of analyses and methods in parasitology and mycology, published in 2018, and the eANOFEL pictures and videos database, freely accessible online (http://www.eanofel.fr). In this article, we draw-up a state-of-the-art of the most specialized techniques available in the parasitology-mycology laboratories and presented in the Catalogue of rare analyses of the Anofel collegium, and their interest for the diagnosis of these infections.