Use of the Mucorales qPCR on blood to screen high-risk hematology patients is associated with better survival.

Our objective was to determine whether the twice-weekly screening of high-risk hematology patients by Mucorales qPCR on serum affects the prognosis of mucormycosis. Results from all serum Mucorales qPCR tests performed on patients from the hematology unit from January 2017 to December 2022 were analyzed. Patients with positive results were classified as having proven, probable or 'PCR-only' mucormycosis. One-month mortality for the local cohort was compared with that of a national cohort of cases of mucormycosis collected by the French surveillance network for invasive fungal disease ('Réseau de surveillances des infections fongiques invasives en France' (RESSIF)) from 2012 to 2018. From 2017 to 2022, 7825 serum Mucorales qPCR tests were performed for patients from the hematology unit; 107 patients with at least one positive Mucorales qPCR (164 positive samples) were identified. Sixty patients (70 positive samples, median Cq = 40) had no radiological criteria for mucormycosis and were considered not to have invasive fungal disease (70/7825, 0.9% false positives). It was not possible to classify disease status for six patients (12 positive samples, median Cq = 38). Forty-one patients (82 positive samples, median Cq = 35) had a final diagnosis of mucormycosis. In comparison with the RESSIF cohort, the local cohort was independently associated with a 48% lower one-month all-cause mortality rate (age-, sex-, and primary disease-adjusted hazard ratio = 0.52; 95% confidence interval: 0.29-0.94; P 0.03). Proactive screening for invasive mold diseases in high-risk hematology patients, including twice-weekly Mucorales qPCR on serum, was associated with mucormycosis higher survival.

Evaluation of Serum Mucorales Polymerase Chain Reaction (PCR) for the Diagnosis of Mucormycoses: The MODIMUCOR Prospective Trial.

BACKGROUND: Early diagnosis and prompt initiation of specific antifungal treatment are essential for improving the prognosis of mucormycosis. We aimed to assess the performance of serum Mucorales quantitative polymerase chain reaction (qPCR) for the early diagnosis and follow-up of mucormycosis. METHODS: We prospectively enrolled 232 patients with suspicion of invasive mold disease, evaluated using standard imaging and mycological procedures. Thirteen additional patients with proven or probable mucormycosis were included to analyze DNA load kinetics. Serum samples were collected twice-a-week for Mucorales qPCR tests targeting the Mucorales genera Lichtheimia, Rhizomucor, and Mucor/Rhizopus. RESULTS: The sensitivity was 85.2%, specificity 89.8%, and positive and negative likelihood ratios 8.3 and 0.17, respectively in this prospective study. The first Mucorales qPCR-positive serum was observed a median of 4 days (interquartile range [IQR], 0-9) before sampling of the first mycological or histological positive specimen and a median of one day (IQR, -2 to 6) before the first imaging was performed. Negativity of Mucorales qPCR within seven days after liposomal-amphotericin B initiation was associated with an 85% lower 30-day mortality rate (adjusted hazard ratio = 0·15, 95% confidence interval [.03-.73], P = .02). CONCLUSIONS: Our study argues for the inclusion of qPCR for the detection of circulating Mucorales DNA for mucormycosis diagnosis and follow-up after treatment initiation. Positive results should be added to the criteria for the consensual definitions from the European Organization for the Research and Treatment of Cancer/Mycoses Study Group Education and Research Consortium (EORTC/MSGERC), as already done for Aspergillus PCR.

Innate and adaptive immune responses following PD-L1 blockade in treating chronic murine alveolar echinococcosis.

BACKGROUND: Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) immune checkpoint blockade are efficacious in certain cancer therapies. OBJECTIVES: The present study aimed to provide a picture about the development of innate and adaptive immune responses upon PD-L1 blockade in treating chronic murine AE. METHODS: Immune treatment started at 6 weeks post-E. multilocularis infection, and was maintained for 8 weeks with twice per week anti-PD-L1 administration (intraperitoneal). The study included an outgroup-control with mice perorally medicated with albendazole 5 d/wk, and another one with both treatments combined. Assessment of treatment efficacy was based on determining parasite weight, innate and adaptive immune cell profiles, histopathology and liver tissue cytokine levels. RESULTS/CONCLUSIONS: Findings showed that the parasite load was significantly reduced in response to PD-L1 blockade, and this blockade (a) contributed to T-cell activity by increasing CD4+ /CD8+ effector T cells, and decreasing Tregs; (b) had the capacity to restore DCs and Kupffer cells/Macrophages; (c) suppressed NKT and NK cells; and thus (d) lead to an improved control of E. multilocularis infection in mice. This study suggests that the PD-L1 pathway plays an important role by regulating adaptive and innate immune cells against E. multilocularis infection, with significant modulation of tissue inflammation.

Investigating new serological and tissue markers for the follow-up of patients operated for alveolar echinococcosis.

AIMS: Alveolar echinococcosis (AE) is characterized by a chronically progressing hepatic injury caused by Echinococcus multilocularis. Surgery presently remains the best curative option. Currently, biological predictive features derived from the resected specimens are not suitable to assess surgery efficacy. The present study was designed to investigate whether a selection of markers measured on the resected specimens exhibits predictive features related to parasite viability, or to a total elimination of the parasite, in addition to serological markers. METHODS AND RESULTS: In a collaboration between two centres, one in France (Besançon), and one in Switzerland (Bern), samples from 40 AE patients were analysed by microarray and serology techniques, individually. Paired serum samples before and after surgery were obtained for 26 patients. In the sera, a significant decrease in PD-L1 levels was observed after surgery, in addition to anti-Em18 levels. In the liver tissue, low levels of Cluster of Differentiation (CD)-3 were correlated with the absence of serum anti-Em18 after surgery. CONCLUSION: This study showed PD-L1 is promising as a potential serological marker and further confirmed the performance of anti-Em18 serology. Further studies on a larger cohort are needed to confirm the utility of performing systematically microarray on resected liver tissue.

Soluble programmed death-1 (sPD-1) as predictor of early surgical outcomes of paediatric cystic echinococcosis.

AIMS: Following treatment, cystic echinococcosis (CE) exhibits a relatively high relapse rate. Here, we evaluated the value of soluble programmed death-1 (sPD-1), sPD-1 ligand (sPD-L1) and anti-recP29 antibody concentrations, as predictors of early surgical treatment outcomes in young CE-affected patients. METHODS AND RESULTS: This prospective study included 59 Tunisian children (177 plasmas), where CE was surgically treated and monitored for 3 post-operative years. Based on CE post-surgical development, patients were clustered into a 'No relapsed' CE (NRCE; n = 39) and a 'Relapsed' CE (RCE; n = 20) group. Plasma levels of sPD-1, sPD-L1 and anti-recP29 IgG were measured using ELISA. In the NRCE group, sPD-1, sPD-L1 and anti-recP29 IgG concentrations were significantly lower at D365 than at D30. By contrast, in the RCE group, no significant difference was observed between D0, D30 and D365. When considering individual variations, the probability to be 'relapse-free' was 67% and 73% when anti-recP29 IgG and sPD-L1 level, respectively, decreased between D30 and D365. The probability to be 'relapse-free' was 86% when the sPD-1 level decreased between D30 and D365 (P = .003; chi-square test). CONCLUSION: sPD-1 may be a useful biomaker for the early evaluation of surgical procedure efficacy in paediatric CE cases.

One year later: The effect of changing azole-treated bulbs for organic tulips bulbs in hospital environment on the azole-resistant Aspergillus fumigatus rate.

Azole-treated plant bulbs have already been evoked as a potential explanation of the worldwide spread of azole-resistant Aspergillus fumigatus (ARAf). We previously pointed out the presence of a high rate of ARAf (71% of A. fumigatus detected on azole-supplemented media) in flower beds containing azole-treated bulbs at the hospital's surroundings. We show here that planting organic bulbs can be a solution to reduce ARAf burden (from 71% rate to below 3%). The results suggest that replacing treated bulbs with organic bulbs may be sufficient to regain a population that is predominantly susceptible in just 1 year. LAY SUMMARY: Antifungal resistance is increasingly observed in fungal pathogens. This study argues that planting organic bulbs in hospitals' outdoor surroundings could be a good alternative to continue to beautify green spaces, without the risk of dissipating antifungal-resistant fungal pathogens.

Echinococcus multilocularis vesicular fluid induces the expression of immune checkpoint proteins in vitro.

AIMS: Alveolar echinococcosis is a severe chronic helminthic infection that mimics a tumour-like disease. This study aimed at investigating in vitro interactions between Echinococcus multilocularis vesicular fluid (VF) and different immune checkpoints (PD-1/PD-L1, CTLA-4, LAG-3 and TIM-3). METHODS AND RESULTS: Peripheral blood mononuclear cells (PBMC) from healthy blood donors were isolated by Ficoll. Natural killer (NK) cells were selected. Each type of cell was stimulated individually with E. multilocularis-VF. Expression of the different immune checkpoints was measured by flow cytometry on day 3 and day 6; all supernatants were used for immunoassays. Cells and supernatants from 22 healthy donors were analysed. A significant increase of PD-1, PD-L1, LAG-3 and TIM-3 was observed upon E. multilocularis-VF exposure for NK cells on day 3 (P < .05, Wilcoxon signed-rank test). A significant increase of PD-L1 and CTLA-4 was observed upon E. multilocularis-VF exposure for T cells on day 6 (P < .05, Wilcoxon signed-rank test), which was associated with increased levels of Th1 and Th2 cytokines P < .05, Wilcoxon signed-rank test). CONCLUSION: These preliminary data suggest that immune checkpoints could be a way for E. multilocularis to modulate the host immune response during alveolar echinococcosis.

Invasive Fungal Disease, Isavuconazole Treatment Failure, and Death in Acute Myeloid Leukemia Patients.

We present 2 fatal cases of invasive fungal disease with isavuconazole treatment failure in immunocompromised patients: one with a TR34-L98H azole-resistant Aspergillus fumigatus isolate and the other a Rhizomucor-A. fumigatus co-infection. Such patients probably require surveillance by galactomannan antigen detection and quantitative PCRs for A. fumigatus and Mucorales fungi.

Survey of 1012 moldy dwellings by culture fungal analysis: Threshold proposal for asthmatic patient management.

Different countries have tried to define guidelines to quantify what levels of fungi are considered as inappropriate for housing. This retrospective study analyzes indoor fungi by cultures of airborne samples from 1012 dwellings. Altogether, 908 patients suffering from rhinitis, conjunctivitis, and asthma were compared to 104 controls free of allergies. Portuguese decree law no 118/2013 (PDL118), ANSES (a French environmental and health agency) recommendations, and health regulations of Besançon University Hospital were applied to determine the rates of non-conforming dwellings, which were respectively 55.2%, 5.2%, and 19%. Environmental microbiological results and medical data were compared. The whole number of colonies per cubic meter of air was correlated with asthma (P < 0.001) and rhinitis (P = 0.002). Sixty-seven genera and species were detected in bedrooms. Asthma was correlated to Aspergillus versicolor (P = 0.004) and Cladosporium spp. (P = 0.02). Thresholds of 300 cfu/m3 for A. versicolor or 495 cfu/m3 for Cladosporium spp. are able to discriminate 90% of the asthmatic dwellings. We propose a new protocol to obtain an optimal cost for indoor fungi surveys, excluding surface analyses, and a new guideline to interpret the results based on >1000 cfu/m3 of whole colonies and/or above threshold levels for A. versicolor or Cladosporium spp.

Storage mite concentrations are underestimated compared to house dust mite concentrations.

Dwellings are increasingly well insulated to save energy and this leads to higher humidity and temperature, which improves conditions for mites. Dermatophagoides antigens are the main allergens involved and tested in atopic asthma. We developed three new species-specific quantitative PCR (qPCR) methods for house dust mites (Dermatophagoides pteronyssinus and D. farinae) and storages mites (Acarus siro, Glycyphagus domesticus, Lepidoglyphus destructor). We sampled dust with electrostatic dust collectors, in the bedrooms, under beds and in the kitchens of patients with allergies (n = 24) and healthy controls (n = 18). Mite quantification was carried out with the three new qPCRs and the qPCR previously described for the Dermatophagoides genus. The qPCRs were highly specific and efficient for house dust mite species and the storage mites. Storage mite concentrations were higher than house dust mite concentrations and were higher in dwellings of patients with allergies. Consequently, allergists should test more often patients against the storage mite antigens by prick tests or IgE serology. Dampness is a major factor in storage mite development and the presence of effective mechanical ventilation can reduce storage mite concentrations four-fold. In addition, to limit exposure to dust mites, treatments should be used throughout dwellings and not only in patients' bedrooms.

Microbial exposure to dairy farmers' dwellings and COPD occurrence.

Dairy farming is a risk factor for chronic obstructive pulmonary disease (COPD). The aim was to determine predictive markers either in blood samples or in dwelling dust samples by comparing COPD and healthy controls with or without farming activity. Dust was collected and analyzed by real-time quantitative PCR. ELISA and DELFIA® were performed to assay the level of specific IgG and IgE of 10 targeted microorganisms. The dwelling exposure of farmers was higher than in the non-farmers (Especially Eurotium amstelodami and Lichtheimia corymbifera). The IgG response against Wallemia sebi and Saccharopolyspora rectivirgula was more often higher in the farmers than the non-farmers. However, exposure and sensitization to the microorganisms tested cannot explain the occurrence of COPD in the dairy farmers' population. COPD development is probably caused by multiple factors associated with exposure over a period of several years.

Quantitative PCR (qPCR) Detection of Mucorales DNA in Bronchoalveolar Lavage Fluid To Diagnose Pulmonary Mucormycosis.

Early diagnosis and treatment are essential to improving the outcome of mucormycosis. The aim of this retrospective study was to assess the contribution of quantitative PCR detection of Mucorales DNA in bronchoalveolar lavage fluids for early diagnosis of pulmonary mucormycosis. Bronchoalveolar lavage fluid samples (n = 450) from 374 patients with pneumonia and immunosuppressive conditions were analyzed using a combination of 3 quantitative PCR assays targeting the main genera involved in mucormycosis in France (Rhizomucor, Mucor/Rhizopus, and Lichtheimia). Among these 374 patients, 24 patients had at least one bronchoalveolar lavage fluid sample with a positive PCR; 23/24 patients had radiological criteria for invasive fungal infections according to consensual criteria; 10 patients had probable or proven mucormycosis, and 13 additional patients had other invasive fungal infections (4 probable aspergillosis, 1 proven fusariosis, and 8 possible invasive fungal infections). Only 2/24 patients with a positive PCR result on a bronchoalveolar lavage fluid sample had a positive Mucorales culture. PCR was also positive on serum in 17/24 patients. In most cases, a positive PCR result was first detected using sera (15/17). However, a positive PCR on bronchoalveolar lavage fluid was the earliest and/or the only biological test revealing mucormycosis in 4 patients with a final diagnosis of probable or proven mucormycosis, 3 patients with probable aspergillosis, and one patient with a possible invasive fungal infection. Mucorales PCR performed on bronchoalveolar lavage fluid could provide additional support for earlier administration of Mucorales-directed antifungal therapy, thus improving the outcome of lung mucormycosis cases.

Immunotherapy of alveolar echinococcosis via PD-1/PD-L1 immune checkpoint blockade in mice.

The growth potential of the tumour-like Echinococcus multilocularis metacestode (causing alveolar echinococcosis, AE) is directly dependent upon the nature/function of the periparasitic adaptive host immune-mediated processes. PD-1/PD-L1 pathway (programmed cell death 1), which inhibits lymphocytic proliferation in tumour development, is over-expressed at the chronic stage of AE. We tested the impact of a PD-1/PD-L1 pathway blockade on the outcome of both chronic AE (intraperitoneal metacestode inoculation, secondary AE and SAE) and acute AE (peroral egg infection, primary AE and PAE). To assess the parasite proliferation potential, we measured parasite mass weight for SAE and liver lesion number for PAE. In both models, the parasite load was significantly decreased in response to anti-PD-L1 antibody treatment. In SAE, anti-PDL1 administration was associated with increased Th1 response parameters and decreased Treg responses, while in PAE anti-PDL1 administration was associated with fewer lesions in the liver and decreased Treg/Th2 responses. Our findings highly suggested that a PD-1/PD-L1 pathway blockade triggered the host immune responses in favour of an immune-mediated control of E. multilocularis proliferation. Based on this, future studies that combine PD-1/PD-L1 blockade with a parasitostatic albendazole medication may yield in a putatively curative therapeutic approach to control alveolar echinococcosis.

Exposure to field vs. storage wheat dust: different consequences on respiratory symptoms and immune response among grain workers.

PURPOSE: The aim of this study was to understand the differential acute effects of two distinct wheat-related dusts, such as field or stored wheat dust handling, on workers' health and how those effects evolved at 6 month intervals. METHODS: Exposure, work-related symptoms, changes in lung function, and blood samples of 81 workers handling wheat and 61 controls were collected during the high exposure season and 6 months after. Specific IgG, IgE, and precipitins against 12 fungi isolated from wheat dust were titrated by enzyme-linked immunosorbent assay, dissociation-enhanced lanthanide fluorescence immunoassay, and electrosyneresis. The level of fungi was determined in the workers' environment. Levels of exhaled fraction of nitrogen monoxide (FENO) and total IgE were obtained. Exposure response associations were investigated by mixed logistic and linear regression models. RESULTS: The recent exposure to field wheat dust was associated with a higher prevalence for five of six self-reported airway symptoms and with a lower FENO than those in the control population. Exposure to stored wheat dust was only associated with cough. No acute impact of exposure on respiratory function was observed. Exposure to field wheat dust led to workers' sensitization against the three field fungi Aureobasidum, Cryptococcus, and Phoma, although exposure to storage wheat dust was associated with tolerance. The level of Ig remained stable 6 months after exposure. CONCLUSION: The clinical picture of workers exposed to field or storage wheat dust differed. The systematic characterization of the aerosol microbial profile may help to understand the reasons for those differences.

Lack of evidence of IgE allergic sensitisation from working with lactic acid bacteria in the dairy foods industry.

This research communication aimed to evaluate the level of immunoglobulin E from lactic acid bacteria (LAB) that are used in dairy industries. Previous studies have demonstrated that workers report symptoms of irritation and are frequently IgG-sensitised to LAB. Workers (n = 44) from a probiotic production unity and the control lab were seen by a medical practitioner and responded to an occupational questionnaire. Specific IgE by the DELFIA® technique against 6 strains of LAB were measured on 44 exposed workers and 31 controls sera. Levels of specific IgE were low and no difference was observed between the two groups. This lack of IgE response could be explained by a healthy worker effect, an efficient implementation of personal protective equipment or by an absence of allergic mechanisms to account for the self-reported irritative symptoms. Despite the high concentrations of LAB, preventive measures are effective enough to guarantee no allergic effect and to prevent other adverse health effects. The implementation of preventive measures to avoid or reduce exposure to dust of LAB, and more generally to milk powder, is recommended in all dairy industry.

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

Echinococcus multilocularis vesicular fluid inhibits activation and proliferation of natural killer cells.

Alveolar echinococcosis is a severe chronic helminthic disease that mimics slow-growing liver cancer. The immune evasion strategy of Echinococcus multilocularis Leuckart, 1863 remains poorly understood. The aim of this study was to investigate in vitro the impact of E. multilocularis vesicular fluid (Em-VF) on peripheral blood mononuclear cells (PBMC) and on natural killer (NK) cells. PBMC and NK cells were exposed to Em-VF (1 µg/ml) during six days. The effect of Em-VF was assessed on CD69, viability and proliferation, and on and transforming growth factor ß (TGF-ß), interferon γ (IFN-γ), interleukin 17 (IL-17) and interleukin 10, using flow cytometry and ELISA, respectively. Exposure to Em-VF had no bearing on PBMC's viability, proliferation and expression of CD69. In contrast, higher levels of IL-17 at day three and of TGF-ß at day six were observed in PBMC supernatant after exposure to Em-VF (p < 0.05, Wilcoxon signed-rank test). Exposure to Em-VF induced a significant decrease of CD69 expression of NK cells at day three and a significant decrease of proliferation of NK cells at day six (p < 0.05, Wilcoxon signed-rank test). In contrast, NK cells viability and levels of cytokines did not vary significantly over Em-VF stimulation. Exposure to Em-VF had a significant bearing on activation and proliferation of NK cells. NK cells may play an important role in the immune response of the host against E. multilocularis.

Development of a Real-Time PCR for a Sensitive One-Step Coprodiagnosis Allowing both the Identification of Carnivore Feces and the Detection of Toxocara spp. and Echinococcus multilocularis.

Studying the environmental occurrence of parasites of concern for humans and animals based on coprosamples is an expanding field of work in epidemiology and the ecology of health. Detecting and quantifying Toxocara spp. and Echinococcus multilocularis, two predominant zoonotic helminths circulating in European carnivores, in feces may help to better target measures for prevention. A rapid, sensitive, and one-step quantitative PCR (qPCR) allowing detection of E. multilocularis and Toxocara spp. was developed in the present study, combined with a host fecal test based on the identification of three carnivores (red fox, dog, and cat) involved in the life cycles of these parasites. A total of 68 coprosamples were collected from identified specimens from Vulpes vulpes, Canis lupus familiaris, Canis lupus, Felis silvestris catus, Meles meles, Martes foina, and Martes martes With DNA coprosamples, real-time PCR was performed in duplex with a qPCR inhibitor control specifically designed for this study. All the coprosample host identifications were confirmed by qPCR combined with sequencing, and parasites were detected and confirmed (E. multilocularis in red foxes and Toxocara cati in cats; 16% of samples presented inhibition). By combining parasite detection and quantification, the host fecal test, and a new qPCR inhibitor control, we created a technique with a high sensitivity that may considerably improve environmental studies of pathogens.

Retrospective study of human cystic echinococcosis over the past decade in France, using a nationwide hospital medical information database.

No specific surveillance system of cystic echinococcosis infections in humans exists in France. The incidence and trends over time have not been documented since the last decade of the twentieth century. We performed the current analysis to assess human cystic echinococcosis epidemiology over a 10-year period in France (including the island of Corsica but excluding the overseas territories) using a nationwide hospital medical information database. A total number of 2629 patients were identified in this database between 2005 and 2014. The average annual incidence rate was 0.42 hospitalized cases/100,000 inhabitants. It was highest in the Island of Corsica (1.76 cases/100,000) and in the region Provence-Alpes-Côte d'Azur (0.85 cases/100,000). This retrospective analysis of hospital records provides a population-based estimate of cystic echinococcosis incidence and trends over a 10-year period at a national level. It indicates a significant decrease of the incidence between 2005 and 2014 at the national level and in the area that reported the highest incidence. It stresses the fact that in France, cystic echinococcosis is not re-emerging and that the incidence remains low in comparison with similar studies in other Mediterranean countries.