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.

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.

Chronic Exposure to Secondary Organic Aerosols Causes Lung Tissue Damage.

Recently, secondary organic aerosols (SOAs) emerged as a predominant component of fine particulate matter. However, the pathogenic mechanism(s) of SOAs are still poorly understood. Herein, we show that chronic exposure of mice to SOAs resulted in lung inflammation and tissue destruction. Histological analyses found lung airspace enlargement associated with massive inflammatory cell recruitment predominated by macrophages. Concomitant with such cell influx, our results found changes in the levels of a series of inflammatory mediators in response to SOA. Interestingly, we observed that the expression of the genes encoding for TNF-α and IL-6 increased significantly after one month of exposure to SOAs; mediators that have been largely documented to play a role in chronic pulmonary inflammatory pathologies. Cell culture studies confirmed these in vivo findings. Of importance as well, our study indicates increased matrix metalloproteinase proteolytic activity suggesting its contribution to lung tissue inflammation and degradation. Our work represents the first in vivo study, which reports that chronic exposure to SOAs leads to lung inflammation and tissue injury. Thus, we hope that these data will foster new studies to enhance our understanding of the underlying pathogenic mechanisms of SOAs and perhaps help in the design of therapeutic strategies against SOA-mediated lung injury.

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.

Polymorphism study of Cryptosporidium hominis gp60 subtypes circulating in Tunisia.

Cryptosporidium spp. are a major cause of gastrointestinal diseases in humans worldwide. While a single subtype of Cryptosporidium hominis has been shown to be responsible for several large outbreaks related to water contamination in developed countries, little is known about the epidemiology of C. hominis in developing countries. This study reports the first genetic characterization of C. hominis at the subtype level in several human populations in Tunisia using the gp60 gene. Eighteen isolates were identified as C. hominis by a restriction fragment length polymorphism (RFLP) analysis. The prevalence of this species in different human populations ranges from 1.53% to 13.04% with a high prevalence being reported in immunocompromised children (13.04%) followed by patients with malignent myeloma (5.5%) and HIV-infected patients (4.59%). The gp60 analysis on C. hominis isolates, performed in 14 cases, showed the presence of a single subtype family: "Ia". Different subtypes were identified within this family (A11G1R1, A12R3, A23G1R1, A26G1R1, A27G1R1, A28G1R1). The IaA26G1R1 subtype was the most dominant subtype described in this area (50%). Despite the high genetic diversity of Cryptosporidium spp, a low heterogeneity at the subtype level was observed within C. hominis circulating in Tunisia. This distribution is an indicator for intensive and stable anthroponotic cryptosporidiosis in this region. Besides, the presence of a unique genotype in 5 HIV-infected patients attending the same hospital ward suggests the possible occurrence of hospital-acquired infection and underlines the need to implement preventive measures to avoid nosocomial transmission.

A cell impedance-based real-time in vitro assay to assess the toxicity of amphotericin B formulations.

Aerosolized liposomal amphotericin B (L-AmB) has been investigated as prophylaxis against invasive aspergillosis. However, the clinical results are controversial and some trials suggest that toxicity could be a limitation for wider use. Our aim was to assess the dynamics of cell toxicity induced in a human alveolar epithelial cell line (A549) after exposure to L-AmB (50 to 400µg/ml) or amphotericin B deoxycholate (D-AmB; 50 to 200µg/ml) by monitoring real-time A549 cell viability using an impedance-based technology. Results were expressed as cell index values integrating cell adhesion, proliferation, and survival. In parallel, the gene expression of proinflammatory cytokines was quantified at 6 and 24h after drug addition by real-time RT-PCR on cell lysates. No sustained reduction of cell indexes was observed with L-AmB or empty liposomes, even at 400µg/ml. Only the highest concentration tested of L-AmB (400µg/ml) yielded transient significant 6-fold and 4-fold induction of TNF-α and IL-8 mRNAs, respectively. In contrast, D-AmB induced a decrease in cell indexes and only the 50µg/ml concentration of D-AmB was followed by cell recovery, higher concentrations leading to cell death. Significant 4-fold, 7-fold and 3-fold inductions of TNF-α, IL-8 and IL-33 mRNAs were also observed at 6h with 50µg/ml of D-AmB. In conclusion, continuous cell impedance measurement showed no toxicity on overall cellular behavior although a slight proinflammatory cytokine expression is possible after L-AmB challenge. Real-time kinetics of cell impedance is an interesting tool for initial screening of cell toxicity.

Diffusion of Pneumocystis jirovecii in the surrounding air of patients with Pneumocystis colonization: frequency and putative risk factors.

In a prospective bicentric study, Pneumocystis jirovecii excretion and diffusion was explored in air samples collected in the rooms occupied by 17 Pneumocystis-colonized patients. P. jirovecii DNA was detected by real-time PCR in the air collected from 3 patients' rooms (17.6%), with identical genotypes in corresponding clinical and air samples. Pneumocystis DNA was detected for 2/3 patients with autoimmune disease treated with corticosteroids versus 1/6 patients with hematologic disease and 0/5 kidney transplant recipients. These data confirm the possible excretion of the fungus by Pneumocystis-colonized patients and thus bring additional arguments for the prevention of airborne transmission in hospital wards.

Molecular diagnosis of toxoplasmosis in immunocompromised patients: a 3-year multicenter retrospective study.

Toxoplasmosis is a life-threatening infection in immunocompromised patients (ICPs). The definitive diagnosis relies on parasite DNA detection, but little is known about the incidence and burden of disease in HIV-negative patients. A 3-year retrospective study was conducted in 15 reference laboratories from the network of the French National Reference Center for Toxoplasmosis, in order to record the frequency of Toxoplasma gondii DNA detection in ICPs and to review the molecular methods used for diagnosis and the prevention measures implemented in transplant patients. During the study period, of 31,640 PCRs performed on samples from ICPs, 610 were positive (323 patients). Blood (n = 337 samples), cerebrospinal fluid (n = 101 samples), and aqueous humor (n = 100 samples) were more frequently positive. Chemoprophylaxis schemes in transplant patients differed between centers. PCR follow-up of allogeneic hematopoietic stem cell transplant (allo-HSCT) patients was implemented in 8/15 centers. Data from 180 patients (13 centers) were further analyzed regarding clinical setting and outcome. Only 68/180 (38%) patients were HIV(+); the remaining 62% consisted of 72 HSCT, 14 solid organ transplant, and 26 miscellaneous immunodeficiency patients. Cerebral toxoplasmosis and disseminated toxoplasmosis were most frequently observed in HIV and transplant patients, respectively. Of 72 allo-HSCT patients with a positive PCR result, 23 were asymptomatic; all were diagnosed in centers performing systematic blood PCR follow-up, and they received specific treatment. Overall survival of allo-HSCT patients at 2 months was better in centers with PCR follow-up than in other centers (P < 0.01). This study provides updated data on the frequency of toxoplasmosis in HIV-negative ICPs and suggests that regular PCR follow-up of allo-HSCT patients could guide preemptive treatment and improve outcome.

Utility of adding Pneumocystis jirovecii DNA detection in nasopharyngeal aspirates in immunocompromised adult patients with febrile pneumonia.

Detection of viral and bacterial DNA in nasopharyngeal aspirates (NPAs) is now a routine practice in emergency cases of febrile pneumonia. We investigated whether Pneumocystis jirovecii DNA could also be detected in these cases by conducting retrospective screening of 324 consecutive NPAs from 324 adult patients (198 or 61% were immunocompromised) admitted with suspected pulmonary infections during the 2012 influenza epidemic season, using a real-time quantitative polymerase chain reaction (PCR) assay (PjqPCR), which targets the P. jirovecii mitochondrial large subunit ribosomal RNA gene. These NPAs had already been tested for 22 respiratory pathogens (18 viruses and 4 bacteria), but we found that 16 NPAs (4.9%) were PjqPCR-positive, making P. jirovecii the fourth most prevalent of the 23 microorganisms in the screen. Eleven of the 16 PjqPCR-positive patients were immunocompromised, and five had underlying pulmonary conditions. Nine NPAs were also positive for another respiratory pathogen. Six had PjqPCR-positive induced sputa less than 3 days after the NPA procedure, and five were diagnosed with pneumocystis pneumonia (four with chronic lymphoproliferative disorders and one AIDS patient). In all six available pairs quantification of P. jirovecii DNA showed fewer copies in NPA than in induced sputum and three PjqPCR-negative NPAs corresponded to PjqPCR-positive bronchoalveolar lavage fluids, underscoring the fact that a negative PjqPCR screen does not exclude a diagnosis of pneumocystosis. Including P. jirovecii DNA detection to the panel of microorganisms included in screening tests used for febrile pneumonia may encourage additional investigations or support use of anti-pneumocystis pneumonia prophylaxis in immunocompromised patients.

Use and limits of (1-3)-ß-d-glucan assay (Fungitell), compared to galactomannan determination (Platelia Aspergillus), for diagnosis of invasive aspergillosis.

This study was undertaken to examine the performance of the Fungitell ß-glucan (BG) assay, to compare it with that of the galactomannan (GM) test for the diagnosis of invasive aspergillosis (IA) in patients with hematological malignancies, and to examine the rates of false-positive BG and GM test results due to ß-lactam antibiotics among sera of patients with Gram-positive or Gram-negative bacteremia and selected sera with false-positive results from the GM test. Serum samples from 105 patients with proven (n = 14) or probable (n = 91) IA, 97 hematology patients at risk for invasive fungal infections, 50 healthy blood donors, and 60 patients with bacteremia were used to study the sensitivities and specificities of the assays. The GM test was more specific than the BG assay (97% versus 82%, respectively; P = 0.0001) and the BG assay was more sensitive than the GM test (81% versus 49%, respectively; P < 0.0001) for IA diagnosis. The study of 49 separate batches of ß-lactam antibiotics showed high and very similar rates of false-positive results for the GM and BG assays (29 and 33%, respectively; P = 0.82) but with an almost complete lack of concordance between the 2 assays. For patients with bacteremia, the rate of false-positive results was much higher with the BG test than with the GM test (37% versus 2%, respectively; P < 0.0001), with no significant difference between Gram-positive and Gram-negative bacteremia. In conclusion, the BG test may be useful for the diagnosis of IA because of its high sensitivity in comparison with the GM test, but the overall benefit of this assay remains limited because of its inadequate specificity and its cost.

Efficacy of liposomal amphotericin B for prophylaxis of acute or reactivation models of invasive pulmonary aspergillosis.

The efficacy of antifungal prophylaxis for prevention of invasive aspergillosis (IA) may depend on whether IA results from recent inhalation of spores or reactivation of latent colonisation. Compare the efficacy of liposomal amphotericin B (LAmB) for prophylaxis in acute and reactivation models of IA. In the acute model, mice immunosuppressed from day 0 were challenged at day 3 with an aerosol of Aspergillus fumigatus. LAmB (15 mg kg(-1) ) was administered at day 0 or at challenge. In the reactivation model, naïve mice exposed to A. fumigatus remained untreated until clearance of spores from the lungs, then immunosuppressed to induce reactivation. A single LAmB dose was administered at start of immunosuppression. In the acute model, a single administration of LAmB at start of immunosuppression was not effective, but an additional administration resulted in a significant decrease in lung fungal burden (P < 0.05 vs. controls). A significant prophylactic efficacy was observed when LAmB was administered once at challenge (P < 0.01). In the reactivation model, a single LAmB administration at start of immunosuppression significantly reduced both reactivation rate and fungal burden vs. controls (P < 0.01). Our results show that the conditions under which IA develop and timing of administration of LAmB were determinant variables for prophylactic efficacy.

Bayesian development of a dose-response model for Aspergillus fumigatus and invasive aspergillosis.

Invasive aspergillosis (IA) is a major cause of mortality in immunocompromized hosts, most often consecutive to the inhalation of spores of Aspergillus. However, the relationship between Aspergillus concentration in the air and probability of IA is not quantitatively known. In this study, this relationship was examined in a murine model of IA. Immunosuppressed Balb/c mice were exposed for 60 minutes at day 0 to an aerosol of A. fumigatus spores (Af293 strain). At day 10, IA was assessed in mice by quantitative culture of the lungs and galactomannan dosage. Fifteen separate nebulizations with varying spore concentrations were performed. Rates of IA ranged from 0% to 100% according to spore concentrations. The dose-response relationship between probability of infection and spore exposure was approximated using the exponential model and the more flexible beta-Poisson model. Prior distributions of the parameters of the models were proposed then updated with data in a Bayesian framework. Both models yielded close median dose-responses of the posterior distributions for the main parameter of the model, but with different dispersions, either when the exposure dose was the concentration in the nebulized suspension or was the estimated quantity of spores inhaled by a mouse during the experiment. The median quantity of inhaled spores that infected 50% of mice was estimated at 1.8 × 10(4) and 3.2 × 10(4) viable spores in the exponential and beta-Poisson models, respectively. This study provides dose-response parameters for quantitative assessment of the relationship between airborne exposure to the reference A. fumigatus strain and probability of IA in immunocompromized hosts.

Weight loss and abnormal lung inflammation in mice chronically exposed to secondary organic aerosols.

Secondary organic aerosols (SOAs) have emerged recently as a major component of fine particulate matter. Cell culture studies revealed a role for SOAs in cell oxidative stress, toxicity and inflammation and only a few studies investigated short-term SOA exposure in animal models. Here, mice were chronically exposed to naphthalene-derived SOAs for one and two months. Weight monitoring indicated a marked mass loss, especially in females, following chronic exposure to SOAs. Significantly, a cytokine antibody microarray approach revealed SOA-induced abnormal lung inflammation similar to that seen in cigarette smoke-induced chronic obstructive pulmonary disease (COPD). This in vivo study testifies to the pathogenic role of sub-chronic SOA exposure on human health.

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.

A 10-year retrospective analysis of Toxoplasma gondii qPCR screening in allogeneic hematopoietic stem cell transplantation recipients.

Weekly blood Toxoplasma gondii DNA screening using real-time quantitative polymerase chain reaction (qPCR) has been implemented in all allogeneic hematopoietic stem cell transplantation (alloHSCT) recipients at our hospital. We retrospectively analyzed the consequences of a positive blood qPCR in the management of Toxoplasma infection (TI) and disease (TD).From 2011 to 2020, 52 (4.13%) of 1 257 alloHSCT recipients had at least one positive qPCR, 45 (3.5%) with TI and seven (0.56%) with TD (central nervous system involvement). Forty-four patients were qPCR-positive before day 100, 30 without and 14 with anti-Toxoplasma prophylaxis. Twenty-five of them (56.8%) started or continued prophylactic dosage treatment: all became qPCR-negative, including 20 (80%) receiving only prophylactic dosage treatment. Twenty-four of them (54.5%) received non-prophylactic dosage treatment: qPCR became negative in 22/24 (91.7%), while TI contributed to death in two cases. Six of the eight patients diagnosed after D100 had breakthrough TI or TD. No death was attributable to TI or TD. qPCR kinetics available for 24 patients increased until anti-Toxoplasma treatment began, then decreased with all treatment regimens.Clinical follow-up and qPCR monitoring with quantification of the parasitic load appears a reasonable strategy to avoid TD and to use minimal effective dosage of anti-Toxoplasma treatments.

Prospective evaluation of clinical and biological markers to predict the outcome of invasive pulmonary aspergillosis in hematological patients.

Early evaluation of treatment efficacy in invasive aspergillosis (IA), a leading cause of morbidity and mortality in hematological patients, remains a challenge. We conducted a prospective study to evaluate the performance of different markers in predicting the outcome of patients with IA. Both clinical and biological criteria were assessed 7, 14, 21, and 45 days after inclusion in the study, and mortality was assessed at day 60. The association between baseline data and their evolution and the day 45 response to treatment was analyzed. A total of 57 patients (4 with proven, 44 with probable, and 9 with possible aspergillosis according to the revised EORTC/MSG [European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and National Institute of Allergy and Infectious Diseases Mycoses Study Group] definitions) were included. At day 45, 30 patients (53%) were determined to be responders, 25 (44%) were nonresponders, and 2 were not able to be evaluated. Twenty patients died within the 60 days of follow-up. We found that a poor day 45 outcome was associated with patients who had high baseline serum galactomannan (GM) antigen levels and those receiving steroids at the time of IA. A consistently negative serum GM index was associated with a good outcome, and the day 14 clinical evaluation was predictive of the day 45 outcome. No association was found between Aspergillus antibodies or DNA detection and patients' outcome. We conclude that the GM index value at diagnosis of IA, GM index kinetics, and clinical evaluation at day 14 are good markers for predicting the outcome of patients with IA and should be taken into account for adapting antifungal treatment.

Dynamics of Pneumocystis carinii air shedding during experimental pneumocystosis.

To better understand the diffusion of Pneumocystis in the environment, airborne shedding of Pneumocystis carinii in the surrounding air of experimentally infected rats was quantified by means of a real-time polymerase chain reaction assay, in parallel with the kinetics of P. carinii loads in their lungs. P. carinii DNA was detected in the air 1 week after infection and increased until 4-5 weeks after infection before stabilizing. A significant correlation was shown between lung burdens and the corresponding airborne levels, suggesting the possibility of estimating the fungal lung involvement through quantification of Pneumocystis in the exhaled air.

Practical checklist for implementation of antifungal stewardship programmes.

Introduction. Antifungal stewardship programmes are needed in healthcare facilities to limit the overuse or misuse of antifungals, which are responsible for an increase in antifungal resistance.Hypothesis/Gap Statement. Core recommendations for antifungal stewardship were published by the Mycoses Study Group Education and Research Consortium, while the Centers for Disease Control and Prevention (CDC) provided a Core Elements of Hospital Antibiotic Stewardship Programs checklist. The recommendations offer global core elements for best practices in antifungal stewardship, but do not provide a framework for the implementation of antifungal stewardship programmes in healthcare facilities.Aim. In line with the recommendations, it is of the utmost importance to establish a practical checklist that may be used to implement antifungal stewardship programmes.Methodology. The practical checklist was established by a national consensus panel of experts involved in antifungal stewardship activities. A preliminary checklist was sent to all experts. The final document was approved by the panel after discussion and the resolution of any disagreements by consensus.Results. The final checklist includes the following items: leadership support; actions to support optimal antifungal use; actions to monitor antifungal prescribing, use and resistance; and an education programme.Conclusion. This antifungal stewardship checklist offers opportunities for antifungal resistance containment, given that antifungal stewardship activities promote the optimal use of antifungals.