Whispers of change in preschool asthma phenotypes: Findings in the French ELFE cohort.

RATIONALE: Early life asthma phenotyping remains an unmet need in pediatric asthma. In France, severe pediatric asthma phenotyping has been done extensively; however, phenotypes in the general population remain underexplored. Based on the course and severity of respiratory/allergic symptoms, we aimed to identify and characterize early life wheeze profiles and asthma phenotypes in the general population. METHODS: ELFE is a general population based birth cohort; which recruited 18,329 newborns in 2011, from 320 maternity units nationwide. Data was collected using parental responses to modified versions of ISAAC questionnaire on eczema, rhinitis, food allergy, cough, wheezing, dyspnoea and sleep disturbance due to wheezing at 3 time points: post-natal (2 months), infancy (age 1) and pre-school (age 5). We built a supervised trajectory for wheeze profiles and an unsupervised approach was used for asthma phenotypes. Chi squared (χ2) test or fisher's exact test was used as appropriate (p < 0.05). RESULTS: Wheeze profiles and asthma phenotypes were ascertained at age 5. Supervised wheeze trajectory of 9161 children resulted in 4 wheeze profiles: Persistent (0.8%), Transient (12.1%), Incident wheezers at age 5 (13.3%) and Non wheezers (73.9%). While 9517 children in unsupervised clusters displayed 4 distinct asthma phenotypes: Mildly symptomatic (70%), Post-natal bronchiolitis with persistent rhinitis (10.2%), Severe early asthma (16.9%) and Early persistent atopy with late onset severe wheeze (2.9%). CONCLUSION: We successfully determined early life wheeze profiles and asthma phenotypes in the general population of France.

Development of a cockroach (Blattella germanica) qPCR for the objective measurement of exposure at home.

Cockroach allergens have a greater impact on asthma morbidity than those from dust mites, cats, and dogs. The American cockroach (Periplaneta americana) and the German cockroach (Blattella germanica) are most frequently responsible for sensitization. The worldwide prevalence of allergic sensitization has been estimated at 2 to 26 % and is influenced by unfavorable socioeconomic conditions. Exposure is generally measured by determining antigen levels in dust or through insect trapping. We developed a real-time quantitative PCR (qPCR) method to provide an objective measurement of B. germanica levels in dwellings. The specificity of the qPCR primers and TaqMan® hydrolysis probe was validated in silico with 18S rRNA sequences. No amplification was observed for other species of cockroaches, with the exception of Blattella nipponica, which is not common indoors. From 2018 to 2021, exposure to B. germanica was detected and quantified in 27 of 389 dwellings in Bourgogne-Franche-Comté (mean = 333.8; median = 9.1 and maximum = 5304 copy number equivalents) and in 236 of 3193 ELFE cohort dwellings in mainland France in 2011 (mean = 15.6; median < 1 and maximum = 1275 copy number equivalents). The distribution of dwellings testing positive for cockroaches (7 %) differed among the 12 regions of France: <1 % in two regions, between 1 and 5 % in eight regions, 16.5 % in two regions and 35 % around Paris. Exposure measurements by the EDC sampling and qPCR methods are effective ways to assess the exposure to cockroaches in dwellings. A knowledge of the level of exposure to cockroaches is particularly important for asthmatic patients, particularly those not allergic to other common antigens.

Bird fancier's lung serodiagnosis by automated r-IgLL1 ELISA.

CONTEXT: Bird fancier's lung (BFL) is the most prevalent form of hypersensitivity pneumonitis (HP) worldwide. The current techniques used for the serological diagnosis of BFL all use crude extracts from feathers, droppings, and blooms as test antigens, which is associated with a lack of standardization and variability of the results. An antigenic protein, immunoglobulin lambda-like polypeptide-1 (IgLL1), isolated from pigeon droppings, was recently identified to be associated with BFL. We used genetic engineering to produce IgLL1 as a recombinant antigen. AIM: We aimed to prospectively validate the use of an automated ELISA based on recombinant IgLL1 protein (r-IgLL1) as the test antigen for the serological diagnosis of BFL. METHODS: Immunoprecipitation (IP) techniques (immunodiffusion (ID), immunoelectrophoresis (IEP)) and ELISA using r-IgLL1 were performed concomitantly over 10 months on 634 sera from patients with a BFL serodiagnosis request. Questionnaires were sent to obtain details on the avian exposure, clinical data, and final diagnosis. Concordance, sensitivity (Se), and specificity (Sp) of the two techniques were compared. RESULTS: In total, 72 completed questionnaires were returned with 18 cases of BFL diagnosed and 54 of non-BFL. The concordance between the ELISA and ID+IEP precipitation techniques was 71%. The combination of immunoprecipitation techniques showed a Se of 78% and a Sp of 67%. The ELISA using r-IgLL1 showed a Se of 89% and a Sp of 91%. The automated r-IgLL1 ELISA test is sufficiently efficient to be used alone for the diagnosis of patients exposed solely to Columbidae. In cases of other avian exposure, the Se and Sp of the r-IgLL1 ELISA used for screening combined with the immunodiffusion test for confirmation were 89% and 93%, respectively. CONCLUSIONS: The automated ELISA using r-IgLL1 is a promising tool for BFL serodiagnosis. Replacing immunodiffusion by the automated ELISA using r-IgLL1 as a screening technique will be the basis of our future strategy for BFL serodiagnosis.

Pilot Study Using Recombinant Antigens r-PROE and r-IGLL1 for the Serodiagnosis of Feather Duvet Lung.

BACKGROUND: Feather duvet lung (FDL) is an underestimated form of acute and chronic hypersensitivity pneumonitis. Serological tests for FDL need to be validated. We investigated the ability of recombinant pigeon Proproteinase E (r-PROE) and Immunoglobulin-lambda-like-polypeptide-1 (r-IGLL1) proteins to support the serological diagnosis of FDL, and propose them as a serological tool for clinicians to differentiate cases from FDL and Bird fancier's lung (BFL). METHODS: Specific IgG antibodies against r-PROE and r-IGLL1, analyzed with ELISA, were measured in patients diagnosed with FDL (n=31), BFL (n=15) controls exposed (n=15) and unexposed to feathers (n=15). RESULTS: The sensitivity and specificity of the r-PROE ELISA for the serological diagnosis of FDL cases versus exposed and unexposed controls were 74.2% and 86.7% respectively, with an index threshold of 0.5 (AUC: 0.89). In addition, this serological test was effective to support the serological diagnosis of FDL and BFL cases with significantly different thresholds. The r-IGLL1 ELISA was only effective for the serological diagnosis of BFL. Also, these two serological tests were useful for the diagnosis of both chronic and acute forms. CONCLUSIONS: The new diagnostic test for FDL using r-PROE protein should help to detect overt and hidden cases of FDL. The combination of both test will help the clinician in distinguish between the etiology of birds or feathers duvet.

Factors associated with Pneumocystis colonization and circulating genotypes in chronic obstructive pulmonary disease patients with acute exacerbation or at stable state and their homes.

Pneumocystis jirovecii colonization is frequent during chronic obstructive pulmonary disease (COPD) and patients constitute potential contributors to its interhuman circulation. However, the existence of an environmental reservoir cannot be excluded. We assessed the prevalence and factors associated with Pneumocystis colonization during COPD, and studied circulation between patients and their domestic environment. Pneumocystis molecular detection and mtLSU genotyping were performed in oro-pharyngeal washes (OPW) sampled in 58 patients with COPD acute exacerbation, and in indoor dust, sampled in patients' homes using electrostatic dust collectors (EDCs). Lung and systemic inflammation was assessed. Pneumocystis carriage was evaluated in 28 patients after 18 months at stable state. Pneumocystis was detected in 11/58 OPWs during exacerbation (19.0%). Colonized patients presented a significantly lower body mass index, and higher serum IL-17 and CD62P. One patient presented positive detection of typable isolates in both OPW and EDC, with both isolates harboring mtLSU genotype 3. Pneumocystis genotype 1 was further detected in EDCs from three non-colonized patients and one colonized patient with non-typable isolate. Genotypes 1 and 2 were predominant in clinical isolates (both 42%), with genotype 3 representing 16% of isolates. Pneumocystis was detected in 3/28 patients at stable state (10.7%). These data suggest that Pneumocystis colonization could be facilitated by a lower BMI and be related to acute alteration of lung function during COPD exacerbation. It also suggests Th17 pathway and platelet activation could be involved in the anti-Pneumocystis response during colonization. Last, Pneumocystis detection in EDCs supports its potential persistence in indoor dust. LAY SUMMARY: Chronic obstructive pulmonary disease patients tend to be more frequently colonized by Pneumocystis during exacerbation (19.0%) than at stable state (10.7%). Factors associated with colonization include lower BMI, higher IL-17, and CD62P. Pneumocystis detection in patients' dwellings suggests potential persistence in indoor dust.

Pilot Study Using Recombinant Antigens r-PROE and r-IGLL1 for the Serodiagnosis of Feather Duvet Lung.

BACKGROUND: Feather duvet lung (FDL) is an underestimated form of acute and chronic hypersensitivity pneumonitis. Serological tests for FDL need to be validated. We investigated the ability of recombinant pigeon Proproteinase E (r-PROE) and Immunoglobulin-lambda-like-polypeptide-1 (r-IGLL1) proteins to support the serological diagnosis of FDL, and propose them as a serological tool for clinicians to differentiate cases from FDL and Bird fancier's lung (BFL). METHODS: Specific IgG antibodies against r-PROE and r-IGLL1, analyzed with ELISA, were measured in patients diagnosed with FDL (n=31), BFL (n=15) controls exposed (n=15) and unexposed to feathers (n=15). RESULTS: The sensitivity and specificity of the r-PROE ELISA for the serological diagnosis of FDL cases versus exposed and unexposed controls were 74.2% and 86.7% respectively, with an index threshold of 0.5 (AUC: 0.89). In addition, this serological test was effective to support the serological diagnosis of FDL and BFL cases with significantly different thresholds. The r-IGLL1 ELISA was only effective for the serological diagnosis of BFL. Also, these two serological tests were useful for the diagnosis of both chronic and acute forms. CONCLUSIONS: The new diagnostic test for FDL using r-PROE protein should help to detect overt and hidden cases of FDL. The combination of both test will help the clinician in distinguish between the etiology of birds or feathers duvet.

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.

Impact of domestic mould exposure on Aspergillus biomarkers and lung function in patients with chronic obstructive pulmonary disease.

Patients with chronic obstructive pulmonary disease (COPD) are frequently colonised or sensitised by Aspergillus, but clinical significance remains unclear. Furthermore, little is known on the impact of indoor mould exposure during COPD. In this study, we assessed the relationship between domestic mould exposure, Aspergillus biomarkers and COPD severity during acute exacerbation and at stable state. Aspergillus section Fumigati culture in sputum and anti-Aspergillus antibodies detection (IgG and precipitins) were followed up in COPD patients that were prospectively recruited during exacerbation (n = 62), and underwent a visit at stable state after 18 months (n = 33). Clinical characteristics were collected at inclusion. Electrostatic dust collectors (EDCs) were used to measure domestic mould contamination. Aspergillus section Fumigati was more frequently detected during exacerbation (16.9%) than at stable state (4.0%), but the frequency of patients presenting with anti-Aspergillus antibodies was similar (32.2% and 33.3%, respectively). Aspergillus section Fumigati detection was associated with a higher body-mass index (BMI) during exacerbation, whereas patients with anti-Aspergillus antibodies presented a lower BMI and forced expiratory volume in 1 s, as well as a higher frequency of inhaled corticoids and higher total mould and Penicillium exposure at final visit (P < 0.05). The frequency of patients with anti-Aspergillus antibodies was higher for total mould counts >30 CFU/cm2 (P = 0.03). Aspergillosis was diagnosed in 2 patients (6.1%) who presented increased levels of antibodies. Our data suggest that anti-Aspergillus antibodies are associated with chronic lung function alteration and/or domestic mould exposure, thereby supporting the consideration of indoor mould contamination and anti-Aspergillus antibodies kinetics in COPD management.

Molecular Epidemiology of Azole-Resistant Aspergillus fumigatus in Sawmills of Eastern France by Microsatellite Genotyping.

Background: Wood chipping has been described as a potential hotspot for the selection of azole-resistant Aspergillus fumigatus (ARAf). We previously reported ARAf isolates in sawmills (Eastern France), most of which contained the TR34/L98H mutation. Methods: To study genotypic relatedness, microsatellite genotyping (short tandem repeat for A. fumigatus (STRAf)) was performed on 41 azole-susceptible A. fumigatus (ASAf) and 23 ARAf isolated from 18 sawmills and two clinical A. fumigatus (sensitive and resistant) isolated from a sinus sample of a woodworker. Results: Fifty-four unique multilocus genotypes (MLGs) were described among the 66 isolates: 13/24 ARAf and 41/42 ASAf. Allelic diversity was higher for ASAf than for ARAf. Among the 24 ARAf, five isolates had their own MLGs. Thirteen ARAf (54%) belonged to the same group, composed of four close MLGs, defined using Bruvo's distance. Thirty-two of the 42 ASAf (76%) had their own MLGs and could not be grouped with the Bruvo's distance cutoff used (0.2). Conclusion: Thus, at a regional scale and in the particular environment of the wood industry, common but also different distinct genotypes, even in the same sawmill, were identified. This suggests that the hypothesis of ARAf clonal expansion from a common strain is probably insufficient to explain genotype emergence and distribution.

Indoor Microbiome: Quantification of Exposure and Association with Geographical Location, Meteorological Factors, and Land Use in France.

The indoor microbial community is a mixture of microorganisms resulting from outdoor ecosystems that seed the built environment. However, the biogeography of the indoor microbial community is still inadequately studied. Dust from more than 3000 dwellings across France was analyzed by qPCR using 17 targets: 10 molds, 3 bacteria groups, and 4 mites. Thus, the first spatial description of the main indoor microbial allergens on the French territory, in relation with biogeographical factors influencing the distribution of microorganisms, was realized in this study. Ten microorganisms out of 17 exhibited increasing abundance profiles across the country: Five microorganisms (Dermatophagoïdes pteronyssinus, Dermatophagoïdes spp., Streptomyces spp., Cladosporium sphaerospermum, Epicoccum nigrum) from northeast to southwest, two (Cryptococcus spp., Alternaria alternata) from northwest to southeast, Mycobacteria from east to west, Aspergillus fumigatus from south to north, and Penicillium chrysogenum from south to northeast. These geographical patterns were partly linked to climate and land cover. Multivariate analysis showed that composition of communities seemed to depend on landscapes, with species related to closed and rather cold and humid landscapes (forests, located in the northeast) and others to more open, hot, and dry landscapes (herbaceous and coastal regions, located in the west). This study highlights the importance of geographical location and outdoor factors that shape communities. In order to study the effect of microorganisms on human health (allergic diseases in particular), it is important to identify biogeographic factors that structure microbial communities on large spatial scales and to quantify the exposure with quantitative tools, such as the multi-qPCR approach.

Effects of plant features on symptoms and airway inflammation in compost workers followed over 18 months.

This study investigated the plant features associated with increased irritation symptoms and levels of inflammation markers among compost workers (CWs). Ninety CWs were followed over 18 months, using questionnaires on respiratory symptoms, fractional exhaled nitric oxide measurements, spirometry, a methacholine bronchial challenge test, and quantification of specific immunoglobulins E (IgE) and G. CWs in plants processing the highest quantities of waste exhibited more airway irritation symptoms. So did the CWs in partially and fully indoor plants as compared to those in plants entirely outdoors. Working in sewage sludge versus green waste plants and having a high level of exposure were associated with higher levels of different IgE. The duration of employment decreased the FEV1 by 16 ml per year. Working in an indoor plant is linked to symptoms and inflammation markers in CWs.

Usefulness of bronchoalveolar lavage in a French pediatric cohort with hypersensitivity pneumonitis.

BACKGROUND: Hypersensitivity pneumonitis (HP) is a rare interstitial lung disease in children, and very little data are available on the frequency, diagnosis, and outcomes of HP. In a pediatric cohort with HP, the characteristics of the CD4/CD8 lymphocyte ratio are often described as nonspecific. METHODS: We used the National French Database (RespiRare) to collect data from the last decade on HP. The diagnosis of HP was defined by the presence of a relevant exposure, clinical symptoms, and compatible lung imaging radiology and was usually defined by positive precipitins antibodies. RESULTS: A total of 16 children with a mean age of 10 years (4-13) presented with HP. All children presented with dyspnea on exertion. Diffuse ground-glass opacity was present in all computed tomography (CT) scans. Research guided by a questionnaire and precipitins antibodies against the corresponding antigens showed that patients were positive for contact with birds with or without fungi. Bronchoalveolar lavage (BAL) was performed in 12 children. The total cell counts were elevated in BAL fluid, with a mean value of 36% lymphocytes. The CD4/CD8 lymphocyte ratio was below one for all children. CONCLUSION: BAL in our pediatric cohort with HP had the same characteristics as that of adults with HP. An HP diagnosis must be considered when dyspnea on exertion and diffuse ground-glass opacity are observed. Carrying out BAL and serological tests can help diagnose and avoid lung biopsy.

Azole-resistant Aspergillus fumigatus in the hospital: Surveillance from flower beds to corridors.

Screening has been performed for azole-resistant Aspergillus fumigatus in the indoor air of the hospital since 2015 and in soil and dust samples since January 2019. In total, 83 azole-resistant A fumigatus isolates with a TR34/L98H mutation have been obtained: 1 from the air of the intensive care unit, 16 from the main corridors, 59 from pots of tulips imported from the Netherlands, and 5 from the soil of trees grown in pots.

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.

Hypersensitivity pneumonitis: A new strategy for serodiagnosis and environmental surveys.

We propose a strategy for serodiagnosis of hypersensitivity pneumonitis (HP): 1) question patients about their private or occupational activity, or visit him on site; 2) select panels of six somatic specific antigens appropriate for each type of exposure; 3) and use ELISA to test concomitantly two recombinant antigens highly specific to Farmer's lung, Metalworking-fluid HP, and for Bird fancier's lung. The serodiagnosis provides an immunological argument that may complete radiological, functional lung exploration and clinical features; 4) If the serodiagnosis is negative but the suspicion of HP is strong, a microbial analysis of the patient's specific exposure is conducted; 5) "A la carte" antigens are produced from the microorganisms isolated in the patient's environment sample and tested; 6) Finally, the patient may be asked to undergo a specific inhalation challenge with the offending antigens in a safety cabin, or to avoid his usual environment for a few days.

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.

Fungal contamination of wind instruments: Immunological and clinical consequences for musicians.

INTRODUCTION: Playing a wind instrument is an increasingly reported cause of hypersensitivity pneumonitis. However, current knowledge about contamination of wind instruments by fungi and specific fungal sensitization is scarce. Therefore, we aimed: (i) to assess the current prevalence and type of fungal contamination of wind instruments, (ii) to identify potential risk factors associated with instrument contamination, and (iii) to evaluate the prevalence of sensitization to these fungi among musicians. MATERIAL AND METHODS: Musicians from music schools in eastern France and who played a wind instrument were prospectively recruited (NCT01487850). The mouthpiece and the reed of their instrument were sampled to quantify the magnitude and type of fungi. Each subject had a physical examination, a mycological analysis of saliva and a blood sample in search of serum precipitins against the most frequent fungi isolated from instruments. The results were compared with those of 40 healthy non-exposed controls. RESULTS: Forty musicians playing a wind instrument (bassoon, clarinet, oboe, saxophone) were included. (i) 95% of wind instruments were colonized by fungi, mainly with Phoma spp., Penicillium spp. and Rhodotorula mucilaginosa; (ii) absence of systematic drying of the instrument was a main contributing factor; (iii) serum precipitins were significantly more present in the musicians' sera than in control sera and were consistent with the fungi present in their instrument. CONCLUSION: This study reveals a constant and specific fungal contamination among wind reed instruments with a significant sensitization among musicians, pleading in favour of regular instrument cleaning. Physicians should be aware of this possible source of antigenic exposure.

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.

Determination of azole fungal residues in soils and detection of Aspergillus fumigatus-resistant strains in market gardens of Eastern France.

Medical azole antifungals are major compounds used to prevent and to treat invasive aspergillosis (IA). Azole fungicides, called DMI (14-alpha demethylase inhibitors), are also widely used for crop protection and have been reported to be linked to azole-resistant A. fumigatus (aR-Af) development in the environment. The aim of this study was to determine whether or not market gardens that spray DMIs in Eastern France are also affected by the presence of aR-Af. Forty aR-Afs were detected in soils in only two of the four market gardens using DMIs, with 23% (7/30) and 10% (3/30) of soils containing aR-Af. A total of 87.5% of these isolates had the TR34/L98H mutation and 22.5% the TR46/Y121F/T289A mutation on the cyp51A gene. Analyses of residual azole concentrations in soils showed the presence of difenoconazole for up to 2 years after spraying, but only in soils of market gardens where aR-Af was detected. It is very important to identify professional activities that could lead to aR-Af development and to develop preventive measures for at-risk patients living near professional activities using DMIs. We have to better understand why, in some cases, the use of DMI is not linked to aR-Af. Measures should be taken to avoid the use of DMI conferring cross-resistance to preserve the efficiency of human therapeutics.