Field-Crop Soils in Eastern France: Coldspots of Azole-Resistant Aspergillus fumigatus.

Triazole fungicides are widely used to treat fungal pathogens in field crops, but very few studies have investigated whether fields of these crops constitute hotspots of azole resistance in Aspergillus fumigatus. Soil samples were collected from 22 fields in two regions of eastern France and screened for triazole residues and azole-resistant A. fumigatus (ARAf). Real-time quantitative PCR (qPCR) was used to quantify A. fumigatus in these soil samples. All the plots contained tebuconazole at concentrations from 5.5 to 19.1 ng/g of soil, and 5 of the 22 plots also contained epoxiconazole. Only a few fungal isolates were obtained, and no ARAf was detected. A. fumigatus qPCR showed that this fungal species was, on average, 5000 times more common in soil from flowerbeds containing ARAf than in soil from field crops. Thus, field-crop soils do not appear to promote A. fumigatus development, even if treated with azole fungicides, and cannot be considered hotspots of resistance. Indeed, our results suggest that they are instead a coldspot of resistance and highlight how little is known about the ecological niche of this species.

Chronic exposure to tebuconazole alters thyroid hormones and plumage quality in house sparrows (Passer domesticus).

Triazoles belong to a family of fungicides that are ubiquitous in agroecosystems due to their widespread use in crops. Despite their efficiency in controlling fungal diseases, triazoles are also suspected to affect non-target vertebrate species through the disruption of key physiological mechanisms. Most studies so far have focused on aquatic animal models, and the potential impact of triazoles on terrestrial vertebrates has been overlooked despite their relevance as sentinel species of contaminated agroecosystems. Here, we examined the impact of tebuconazole on the thyroid endocrine axis, associated phenotypic traits (plumage quality and body condition) and sperm quality in wild-caught house sparrows (Passer domesticus). We experimentally exposed house sparrows to realistic concentrations of tebuconazole under controlled conditions and tested the impact of this exposure on the levels of thyroid hormones (T3 and T4), feather quality (size and density), body condition and sperm morphology. We found that exposure to tebuconazole caused a significant decrease in T4 levels, suggesting that this azole affects the thyroid endocrine axis, although T3 levels did not differ between control and exposed sparrows. Importantly, we also found that exposed females had an altered plumage structure (larger but less dense feathers) relative to control females. The impact of tebuconazole on body condition was dependent on the duration of exposure and the sex of individuals. Finally, we did not show any effect of exposure to tebuconazole on sperm morphology. Our study demonstrates for the first time that exposure to tebuconazole can alter the thyroid axis of wild birds, impact their plumage quality and potentially affect their body condition. Further endocrine and transcriptomic studies are now needed not only to understand the underlying mechanistic effects of tebuconazole on these variables, but also to further investigate their ultimate consequences on performance (i.e. reproduction and survival).

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.

Refractory invasive pulmonary aspergillosis due to Aspergillus flavus detected with the combination of two in-house Aspergillus qPCR.

We present a case of probable invasive pulmonary aspergillosis due to Aspergillus flavus, in a female patient treated for an acute myeloid leukemia. Two weeks after an allogenic stem cell transplantation a probable invasive pulmonary aspergillosis was diagnosed based on thoracic imaging combined with positive galactomannan antigen and positive in-house mitochondrial Aspergillus qPCR in serum. Although an antifungal treatment was initiated, Aspergillus qPCR and galactomannan antigen remained positive in serum and worsening of the thoracic lesions was observed. The discordance between the negativity of the in-house ribosomal Aspergillus qPCR (specific to A. fumigatus) and the positivity of the in-house mitochondrial Aspergillus qPCR (targeting A. fumigatus and some other Aspergillus) allowed the suspicion of a thermophilic Aspergillus species that was not A. fumigatus. No strain was obtained in culture but the involvement of A. flavus was confirmed using a specific A. flavus qPCR. This case illustrated the usefulness of our original strategy combining two different in-house Aspergillus qPCRs, in addition to galactomannan assay, to diagnose invasive aspergillosis in hematology patients.

Evidence of environmental transfer of tebuconazole to the eggs in the house sparrow (Passer domesticus): An experimental study.

Triazole compounds are among the most widely used fungicides in agroecosystems to protect crops from potential fungal diseases. Many farmland birds spend a significant part of their life cycle in agroecosystems, which may chronically expose them to pesticides. We experimentally tested whether exposure to environmental concentrations of tebuconazole could induce a contamination of the eggs in an agroecosystem sentinel species, the house sparrow (Passer domesticus). Wild-caught adult sparrows were maintained in captivity and exposed (exposed group) or not (control group) for seven months to tebuconazole through drinking water. Eggs were opportunistically collected for the determination of tebuconazole concentration by Liquid Chromatography coupled to tandem Mass Spectrometry in eggs. We found that eggs from exposed parents all contained tebuconazole with a mean concentration of 1.52 ng g-1 dry weight. In eggs from control parents, the tebuconazole concentration was below the limit of quantification (0.23 ng g-1 dry weight) for 11 out of 13 eggs. Thus, our study demonstrates for the first time that environmental exposure of female birds to tebuconazole can translate into egg contamination by this fungicide.

Experimental Exposure to Tebuconazole Affects Metabolism and Body Condition in a Passerine Bird, the House Sparrow (Passer domesticus).

Triazole compounds are among the most widely used fungicides in agroecosystems to protect crops from potential fungal diseases. Triazoles are suspected to have an impact on nontarget species due to their interactions with nonfungal sterol synthesis, and wild birds are likely to be contaminated by triazole fungicides because many of them live in agroecosystems. We experimentally tested whether exposure to environmental concentrations of a triazole could alter key integrative traits (metabolic rates and body condition) of an agroecosystem sentinel species, the house sparrow (Passer domesticus). Wild-caught adult sparrows were maintained in captivity and exposed (exposed group) or not (control group) for 7 continuous months to tebuconazole through drinking water. The metabolic rates of exposed and control sparrows were then measured at two different temperatures (12 °C and 25 °C), which correspond, respectively, to the thermoregulation and thermoneutrality temperatures of this species. We found that exposed sparrows had lower resting metabolic rates (i.e., measured at thermoneutrality, 25 °C) than controls. However, the thermoregulatory metabolic rates (i.e., measured at 12 °C) did not differ between exposed and control sparrows. Although the body mass and condition were not measured at the beginning of the exposure, sparrows at the time of the metabolic measurements 7 months after the onset of such exposure had a higher body condition than controls, supporting further the idea that tebuconazole affects metabolic functions. Our study demonstrates for the first time that the use of tebuconazole can alter metabolism and could potentially lead to adverse effects in birds. Environ Toxicol Chem 2022;41:2500-2511. © 2022 SETAC.

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.

Molecular Epidemiology of Azole-Resistant Aspergillus fumigatus in France Shows Patient and Healthcare Links to Environmentally Occurring Genotypes.

Resistance of the human pathogenic fungus Aspergillus fumigatus to antifungal agents is on the rise. However, links between patient infections, their potential acquisition from local environmental sources, and links to global diversity remain cryptic. Here, we used genotyping analyses using nine microsatellites in A. fumigatus, in order to study patterns of diversity in France. In this study, we genotyped 225 local A. fumigatus isolates, 112 azole susceptible and 113 azole resistant, collected from the Bourgogne-Franche-Comté region (Eastern France) and sampled from both clinical (n = 34) and environmental (n = 191) sources. Azole-resistant clinical isolates (n = 29) were recovered mainly from cystic fibrosis patients and environmental isolates (n = 84) from market gardens and sawmills. In common with previous studies, the TR34/L98H allele predominated and comprised 80% of resistant isolates. The genotypes obtained for these local TR34/L98H isolates were integrated into a broader analysis including all genotypes for which data are available worldwide. We found that dominant local TR34/L98H genotypes were isolated in different sample types at different dates (different patients and types of environments) with hospital air and patient's isolates linked. Therefore, we are not able to rule out the possibility of some nosocomial transmission. We also found genotypes in these same environments to be highly diverse, emphasizing the highly mixed nature of A. fumigatus populations. Identical clonal genotypes were found to occur both in the French Eastern region and in the rest of the world (notably Australia), while others have not yet been observed and could be specific to our region. Our study demonstrates the need to integrate patient, healthcare, and environmental sampling with global databases in order to contextualize the local-scale epidemiology of antifungal resistant aspergillosis.

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.

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.

Positive quantitative PCR detecting Fusarium solani in a case of mixed invasive fungal disease due to Mucorales and Fusarium solani.

We present a case of invasive fungal co-infection in a young patient treated for an acute myeloid leukemia and having undergone a twice-haploid matched unrelated donor hematopoietic stem cell transplantation (HSCT) with two different donors. A mucormycosis diagnosis was made shortly after the patient's admission using imagery and specific Mucorales qPCR which was treated with liposomal amphotericin B and posaconazole. Twenty days later, a blood culture was positive for Fusarium solani, and disseminated cutaneous lesions appeared. The antifungal treatment was changed to liposomal amphotericin B and voriconazole. Thanks to a complete hematological reconstitution and despite a co-infection with two aggressive filamentous opportunistic fungi, the patient recovered. We took advantage of this clinical case to test a specific Fusarium solani qPCR, which proved to be promising when performed retrospectively on some of the patient samples.

Cardiovascular Risk in COPD: Deciphering the Contribution of Tobacco Smoking.

BACKGROUND: The observation that COPD is an independent risk factor for cardiovascular disease (CVDs) comes from comparisons between smokers with COPD and smokers without COPD. The mechanisms that explain increased risk of CVD in patients with COPD are still unclear. OBJECTIVES: The goal of this study was to assess systemic arterial stiffness (a predictor of CVD mortality) and to evaluate its determinants in a group of patients with mild to moderate COPD secondary to organic dust exposure, tobacco smoking, or both. METHODS: Systemic arterial stiffness was assessed by using aortic pulse wave velocity (aPWV). Measurements were made in 142 patients with COPD and 155 healthy control subjects matched for age, sex, BMI, and tobacco smoking, exposed to tobacco smoking (n = 56/70 for COPD/control subjects, respectively), organic dusts (n = 44/48), or both (n = 42/37). RESULTS: aPWV was higher in COPD than in healthy controls in subjects exposed to tobacco smoking and to both organic dusts and tobacco smoking. By contrast, among never smokers exposed to organic dusts, patients with COPD and matched control subjects had similar aPWV. Multivariate analysis of the 142 patients with COPD (exposed to tobacco smoking and/or to organic dusts) showed that tobacco smoking was associated with high aPWV. Moreover, soluble suppression of tumorigenicity 2, a marker of major cardiovascular events, was correlated with aPWV in these patients. CONCLUSIONS: Analysis of an unselected group of patients with COPD with different causes suggests that: (1) COPD by itself is not sufficient to explain increased aPWV; and (2) tobacco smoking is a risk factor for elevated aPWV in COPD.

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.

Hemp to limit diffusion of difenoconazole in vegetable garden soils.

Triazole molecules are used to manage invasive aspergillosis, a fungal infection mainly due to Aspergillus fumigatus. A. fumigatus is not a phytopathogen, but, as it is widespread in soils, triazole fungicides have an unintended impact on it, selecting resistant populations' in environment. Thus, to maintain our ability to control fungal infections, whether in human health or agriculture, reduce the impact of the use of triazoles in the environment is important, notably limiting their diffusion in soils. Here we tested a hemp-based material as adsorbent to limit the spread of difenoconazole, a triazole fungicide, in vegetable soils. We studied the effects of contact time, material dose, difenoconazole concentration, and organic content of the soil using batch mode and percolation methods. Batch experiments showed that the material exhibited high adsorption capacities toward difenoconazole. Removal from the soil water increased from 46.6% using 0.35 g hemp per kg of soil to 77.0% using 1.75 g hemp per kg, for a contact time of 15 min and an initial difenoconazole concentration of 1.2 mg/L. For a contact time of 240 min, the removal was 93.5%. Percolation experiments showed that the quantity of difenoconazole removed was greater than the amount obtained by batch method: 41.9% of removal with only one passes of solution at a concentration of 12 mg/L is obtained through percolation technique whereas, with similar conditions, only 20% of removal is obtained by batch method, i.e. after 1 min of contact. The removal was strongly dependent on the number of passes: the values increased from 57.0% to 91.0% with increasing the number of passes from 1 to 15. Addition of hemp to soils allows to remove efficiently the difenoconazole fungicide from soil water. Hemp-based felt is a new and safe adsorbent that can be applied in agriculture to limit crop contamination.

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.

Dietary Patterns and Prevalence of Post-bronchodilator Airway Obstruction in Dairy Farmers Exposed to Organic Dusts.

Exposure to organic dusts is an independent causative factor of chronic obstructive pulmonary disease (COPD). Unhealthy dietary patterns have been associated with poor lung function in smokers. This study investigated whether dietary patterns were associated with post-bronchodilator airway obstruction, a hallmark of COPD, in dairy farmers exposed to organic dusts. All subjects were identified by screening programs and patients with airflow obstruction were matched with subjects with normal spirometry. Six groups were compared, defined by their exposures (non-smoking dairy farmers, smokers ≥ 10 pack-years with no occupational exposure, and smoking dairy farmers) and the presence or absence of post-bronchodilator airflow obstruction, resulting in 321 study subjects. The Alternative Healthy Eating Index (AHEI) score was calculated based on an adapted food frequency questionnaire. Mean total AHEI scores were similar in all groups. Comparison between smokers with post-bronchodilator airway obstruction and subjects with post-bronchodilator airway obstruction related to occupational exposure found minimal differences in dietary patterns: dairy farmers had lower scores for the ratio of white to red meat and higher scores for cereal fiber consumption. As in previous studies, smokers with post-bronchodilator airway obstruction exhibited higher lipid intakes and lower carbohydrate intakes than their counterparts with normal spirometry. No evidence of any meaningful difference in dietary patterns was found between subjects with post-bronchodilator airway obstruction detected by screening and healthy controls, either in dairy farmers or in smokers with no occupational exposure.

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.

Molecular Strategies to Diagnose Mucormycosis.

Molecular techniques have provided a new understanding of the epidemiology of mucormycosis and improved the diagnosis and therapeutic management of this life-threatening disease. PCR amplification and sequencing were first applied to better identify isolates that were grown from cultures of biopsies or bronchalveolar lavage samples that were collected in patients with Mucorales infection. Subsequently, molecular techniques were used to identify the fungus directly from the infected tissues or from bronchalveolar lavage, and they helped to accurately identify Mucorales fungi in tissue samples when the cultures were negative. However, these tools require invasive sampling (biospsy, bronchalveolar lavage), which is not feasible in patients in poor condition in Hematology or Intensive Care units. Very recently, PCR-based procedures to detect Mucorales DNA in non-invasive samples, such as plasma or serum, have proved successful in diagnosing mucormycosis early in all patients, whatever the clinical status, and these procedures are becoming essential to improving patient outcome.