Investigation of the value of precipitins in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patients with a positive marker for Aspergillus species.

Although a high prevalence of COVID-19-associated pulmonary aspergillosis has been reported, it is still difficult to distinguish between colonization with Aspergillus fumigatus and infection. Concomitantly, similarities between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and hypersensitivity pneumonitis were suggested. The objective of this study was to investigate retrospectively if precipitin assays targeting A. fumigatus could have been useful in the management of SARS-CoV-2 patients hospitalized in an Intensive Care Unit (ICU) in 2020. SARS-CoV-2 ICU patients were screened for Aspergillus co-infection using biomarkers (galactomannan antigen, qPCR) and culture of respiratory samples (tracheal aspirates and bronchoalveolar lavage). For all these patients, clinical data, ICU characteristics and microbial results were collected. Electrosyneresis assays were performed using commercial A. fumigatus somatic and metabolic antigens. ELISA were performed using in-house A. fumigatus purified antigen and recombinant antigens.Our study population consisted of 65 predominantly male patients, with a median ICU stay of 22 days, and a global survival rate of 62%. Thirty-five patients had at least one positive marker for Aspergillus species detection. The number of arcs obtained by electrosyneresis using the somatic A. fumigatus antigen was significantly higher for these 35 SARS-CoV-2 ICU patients (P 0.01, Welch's t-test). Our study showed that SARS-CoV-2 ICU patients with a positive marker for Aspergillus species detection more often presented precipitins towards A. fumigatus. Serology assays could be an additional tool to assess the clinical relevance of the Aspergillus species in respiratory samples of SARS-CoV-2 ICU patients. LAY SUMMARY: This study showed retrospectively that precipitin assays, such as electrosyneresis, could be helpful to distinguish between colonization and infection with Aspergillus fumigatus during the management of severe acute respiratory syndrome Coronavirus-2 (SARS CoV-2) patients in an intensive care unit.

Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Hypersensitivity pneumonitis in a cystic fibrosis patient.

Hypersensitivity pneumonitis (HP) is a chronic inflammatory lung disease caused by repeated inhalation of antigenic substances. We present a case of metalworking fluids (MWFs)-HP sensitized to Pseudomonas oleovorans in a cystic fibrosis patient. This case illustrates that HP diagnosis remains challenging, especially in patients with another pulmonary disease, and that serodiagnosis contributes to identifying the precise microorganism involved. It also demonstrates that P. oleovorans is an important secondary aetiological agent in MWF-HP, less known than Mycobacterium immunogenum.

Azole-resistant Aspergillus fumigatus in sawmills of Eastern France.

AIMS: Emergence of azole-resistant Aspergillus fumigatus complicates management of Aspergillus diseases. Currently, selection pressure caused by azole fungicide use in farming is strongly suspected of creating resistance. As sawmills also use azole fungicides, we investigated the presence of azole-resistant strains in this environment and studied the relationship between azole fungicide use and development of resistance. METHODS AND RESULTS: Air (n = 200) and substrate (n = 600) samples were taken in 20 sawmills. Azole-resistant strains (Etest and EUCAST methods) were confirmed by sequencing the cyp51A gene and its promoters. Dosage of propiconazole and tebuconazole was performed by gas chromatography coupled with mass spectrometry. Twenty-four azole-resistant A. fumigatus strains were collected among 20 of the 600 substrate samples (3%). Eighty-three percent of theses strains had TR34 /L98H mutation. A significantly higher number of resistant strains was collected in sawmills using fungicide products made with propiconazole mixed with a high concentration of tebuconazole (P = 0·009). The presence of resistant strains was significantly linked to propiconazole quantities in substrates (P = 0·03). CONCLUSIONS: The outcome of azole-resistant A. fumigatus carrying TR34 /L98H mutation seems to greatly depend on the azole fungicide formulation and quantities of azole. These preliminary results are valuable to propose new approaches limiting the emergence of azole-resistant strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Azole resistance is an emerging problem in A. fumigatus and threatens clinical advances made possible by the use of azole antifungals in the treatment of Aspergillus-related diseases. Azole fungicides are also used in the wood industry, notably in sawmills, to protect wood from wood-destroying fungi. Through our study, we show that sawmills represent another professional environment affected by the presence of azole-resistant A. fumigatus strains carrying the TR34 /L98H mutation. Moreover, this study provides valuable preliminary results to propose some new approaches to limit the emergence of azole-resistant A. fumigatus strains.

Identification of a new Y chromosome haplogroup in Spanish native cattle.

The aim of this work was to perform a thorough analysis of the diversity of Y-haplotypes in Spanish cattle. A total of 207 Bos taurus males were sampled across 25 European breeds, with a special focus on rare, local Spanish populations. Animals were genotyped with five Y-specific microsatellites (INRA189, UMN0103, UMN0307, BM861 and BYM1), two indels (ZFY10 and USP9Y) and one SNP (UTY19). A new haplogroup, distinct from those described by Götherström et al. (2005), was identified and named Y1.2. Samples representing the three B. taurus Y-haplogroups were genotyped for four additional Y chromosome SNPs (rs121919254, rs121919281, rs121919323 and rs137049553). Among these SNPs, only rs121919281 was informative in B. taurus and helped to confirm the new Y1.2 haplogroup. Analysis of a larger dataset of standardized haplotypes for 1507 individuals from 57 populations from Spain, other European countries and Africa showed the new Y1.2 haplogroup to be found exclusively in Spanish breeds. This finding reinforces the importance of local Spanish cattle as reservoirs of genetic diversity as well as the importance of the Iberian Peninsula in the history of cattle.

Microbiological consequences of indoor composting.

Recycling of organic waste appeals to more and more people. The aim of this study was to evaluate the microbiological contamination around organic waste bins at three distances over a 12-month period. Contamination near the customary trash of control households was evaluated at the beginning to ensure that there is no recruitment bias. Air samples using the MAS 100 impactor were carried out in 38 dwellings that do household waste composting and in 10 dwellings of controls. Collection of particles by CIP 10 rotating cup sampler and dust samples collected by electrostatic dust collector cloths were acquired in dwellings that do household waste composting. Samples were analyzed by culture and by real-time quantitative PCR. Information about dwelling characteristics and inhabitant practices was obtained by a standardized questionnaire. The genera most often isolated were Penicillium, Aspergillus, Cladosporium and Streptomyces. Near the organic waste bins, bioaerosol samples showed an increase of Acarus siro (P = 0.001). Sedimented dust analyses highlighted an increase of A. siro, Wallemia sebi, Aspergillus versicolor, and Cladosporium sphaerospermum concentrations after a 12-month survey compared to the beginning. Composting favors microorganism development over time, but does not seem to have an effect on the bioaerosol levels and the surface microbiota beyond 0.5 m from the waste bin.

Combining Aspergillus mitochondrial and ribosomal QPCR, in addition to galactomannan assay, for early diagnosis of invasive aspergillosis in hematology patients.

The combination of two quantitative Aspergillus PCR assays, targeting a mitochondrial and a ribosomal target (AfQPCR), has proved effective for diagnosing invasive aspergillosis (IA) in hematology patients with risk factors and a positive galactomannan antigen (GM). The aim of the present study was to assess the performance of systematic AfQPCR for IA screening in at risk patients in a hematology intensive care unit (ICU). The study was performed in the hematology ICU at Besançon University Hospital from March 2012 to December 2013. GM detection (Platelia Aspergillus, Biorad, France) and AfQPCR were performed on the same serum sample, twice a week, in all patients with risk factors for IA. Risk factors and clinical, radiological, and biological data were prospectively recorded using the information sheet from the French network for the surveillance of Invasive Fungal Infection. Thirty-two patients were diagnosed with proven, probable, or possible IA according to the 2008 EORTC/MSG criteria. Sixteen patients had a positive AfQPCR: 9/16 had a positive GM at the same time (GM index >0.5), 4/16 had a positive GM before the AfQPCR and 3/16 had a negative GM at the time of the positive AfQPCR. Screening at risk patients using both AfQPCR and GM on the same serum sample is very feasible in a routine clinical setting. Our results confirm the usefulness of combining biomarkers for an early IA diagnosis.

Evaluation of invasive aspergillosis risk of immunocompromised patients alternatively hospitalized in hematology intensive care unit and at home.

UNLABELLED: Contrary to hospital exposure, little is known about the indoor fungal exposure of hematology patients at home. The aim of our study was to investigate the mold exposure of hematology patients both at home and at hospital to assess their invasive aspergillosis (IA) risk. Fungal exposure was assessed by quantifying opportunistic molds at hospital during hospitalization and in homes of 53 hematology patients. IA was diagnosed in 13 of 53 patients and invasive fungal infection (IFI) in one patient. In hospital, no opportunistic species, or low levels of opportunistic species, were found in 98% of weekly controls. Only 2% of hematology intensive care unit (ICU) controls showed a high level of Aspergillus fumigatus spores in corridor air. Five patients IA were hospitalized during these periods. Seven dwellings of 53 (5/14 dwellings of patients with IA/IFI and 2/39 dwellings of non-IA patients) had a percentage of A. fumigatus and Aspergillus flavus to total mold (significant predictor variable of IA/IFI in our study, general linear model, P-value = 0.02) as high as 15%. Maintaining a 'zero Aspergillus' goal at hospital is essential, and establishing specific and individually opportunistic mold monitoring at home could help to further reduce the IA risk through continuous surveillance. PRACTICAL IMPLICATIONS: This study emphasizes the fact that preventive measures should not be aimed only at the hospital setting: among patients diagnosed with invasive aspergillosis/invasive fungal infection (IA/IFI), 5 of 14 (36%) were exposed to opportunistic fungal species at home exclusively. Moreover, four of these five patients were living in homes having the highest percentage of Aspergillus fumigatus and Aspergillus flavus (>15%), one of which had 48% of A. fumigatus. Therefore, our work supports the need for a counselor to carry out an environmental survey in patients' homes.

Molecular diagnosis of polycystic echinococcosis due to Echinococcus vogeli in a Paraguayan immigrant in Argentina.

Polycystic echinococcosis due to Echinococcus vogeli is a rare parasitic infection that occurs in rural areas of Central and South America. Only molecular identification performed on formalin-fixed paraffin-embedded liver tissue samples gave an unequivocal diagnosis of this disease in a Paraguayan immigrant in Argentina.

A high-resolution 15,000(Rad) radiation hybrid panel for the domestic cat.

The current genetic and recombination maps of the cat have fewer than 3,000 markers and a resolution limit greater than 1 Mb. To complement the first-generation domestic cat maps, support higher resolution mapping studies, and aid genome assembly in specific areas as well as in the whole genome, a 15,000(Rad) radiation hybrid (RH) panel for the domestic cat was generated. Fibroblasts from the female Abyssinian cat that was used to generate the cat genomic sequence were fused to a Chinese hamster cell line (A23), producing 150 hybrid lines. The clones were initially characterized using 39 short tandem repeats (STRs) and 1,536 SNP markers. The utility of whole-genome amplification in preserving and extending RH panel DNA was also tested using 10 STR markers; no significant difference in retention was observed. The resolution of the 15,000(Rad) RH panel was established by constructing framework maps across 10 different 1-Mb regions on different feline chromosomes. In these regions, 2-point analysis was used to estimate RH distances, which compared favorably with the estimation of physical distances. The study demonstrates that the 15,000(Rad) RH panel constitutes a powerful tool for constructing high-resolution maps, having an average resolution of 40.1 kb per marker across the ten 1-Mb regions. In addition, the RH panel will complement existing genomic resources for the domestic cat, aid in the accurate re-assemblies of the forthcoming cat genomic sequence, and support cross-species genomic comparisons.

Microbiological evaluation of ten French archives and link to occupational symptoms.

UNLABELLED: Fungi that damage documents in archives may harm workers' health, depending on which mold species are inhaled, the concentrations of fungal species inhaled, and individual factors. Our aim was to identify and quantify fungi in archives and to investigate possible links with the symptoms experienced by workers. Ten French archives were sampled using an air impactor and electrostatic dust collectors. Allergies and general symptoms felt by 144 workers were reported using a self-report questionnaire. Utilizing culture-based analysis methods along with qPCR, Penicillium chrysogenum, Cladosporium sphaerospermum, and Aspergillus versicolor were the three main fungi in air and dust in terms of quantity and frequency. Median fungal concentrations in storage areas, ranged from 30 to 465 CFU/m(3). People working in the most contaminated archives did not report more symptoms of allergy than others. However, workers in contact with moldy documents reported more headaches (odds ratio, 2.4; 95% confidence interval, 1.1-5.3), fatigue (OR, 2.9; 95% CI, 1.2-6.7), eye irritation (OR, 5.4; 95% CI, 1.9-14.9), throat irritation (OR, 2.4; 95% CI, 1.0-5.7), coughing (OR, 3.2; 95% CI, 1.2-8.4), and rhinorrhea (OR, 2.6; 95% CI, 1.0-6.4) than others. Other parameters such as dust levels and concentrations of metabolites and chemical substances should be considered as confounding factors in further investigations to isolate the role of molds. PRACTICAL IMPLICATIONS: Most studies about fungi and archives deal with the conservation of manuscripts and documents, and few discuss workers' health problems. Our study shows that archives do not represent a highly contaminated environment. Symptoms felt by workers were more often linked to direct contact with moldy documents than to high concentrations of mold in the air of archive storage areas. This study provides data on concentration levels in archives that could be used to interpret microbiological investigations in this type of environment in the future.

Hypersensitivity pneumonitis and metalworking fluids contaminated by mycobacteria.

Metalworking fluids (MWF) are responsible for hypersensitivity pneumonitis (HP). The aim of the present study was to identify the antigen (Ag) responsible for MWF-associated HP, and to optimise serological diagnosis by definition of a threshold allowing discrimination between HP patients and asymptomatic exposed workers. 13 patients, who were workers at a car engine manufacturing plant, were suspected of MWF-associated HP. Microbial analysis of 83 used MWFs was carried out. Sera from 13 MWF-associated HP patients, 12 asymptomatic exposed workers and 18 healthy unexposed controls were tested to determine their immunological responses to three Ags, including Mycobacterium immunogenum. M. immunogenum was identified in 40% of used fluids by culture and confirmed by DNA sequencing. The threshold for differentiating MWF-associated HP patients from asymptomatic exposed workers was five arcs of precipitation (sensitivity 77% and specificity 92%), as determined by electrosyneresis (ES). Using ELISA methods with protein extract from M. immunogenum, a threshold leading to 92% sensitivity and 100% specificity was established. The detection of specific antibodies against M. immunogenum Ag at high levels in case sera suggests that M. immunogenum-contaminated MWF is responsible for MWF-associated HP. To discriminate MWF-associated HP patients from asymptomatic exposed workers, we suggest a five-arc threshold for ES and a 1.6-AU threshold for ELISA methods.

Ribosomal and mitochondrial DNA target for real-time PCR diagnosis of invasive aspergillosis.

The aim of the present study was to assess the diagnostic efficacy of a combination of two quantitative Aspergillus PCR assays, targeting a mitochondrial and a ribosomal target, in patients with risk factors for invasive aspergillosis (IA) and positive galactomannan (GM) antigen. Forty-four patients with hematological malignancies and risk factors for IA according to revised European Organization for Research on Treatment of Cancer and the Mycoses Study Group criteria (EORTC/MSG) criteria and presenting at least two sequential GM-positive sera were included in the study. Mitochondrial PCR was carried out prospectively on all GM-positive serum samples. Ribosomal PCR was carried out retrospectively on frozen stored sera. The sensitivities of mitochondrial and ribosomal PCRs were 58% and 50%, respectively. The diagnostic test performance was improved by using a combination of both PCR assays and by considering a patient PCR positive when at least two positive results were obtained. The sensitivity, specificity, and positive and negative likelihood ratios were 65%, 94%, and 11.8 and 0.37, respectively. A significant association between fatal outcome at 90 days and positive results of ribosomal PCR assays was observed (adjusted hazard ratio = 8.2; 95% confidence interval [CI] = 1.0 to 65.8; P = 0.048). Our results showed that the combination of two PCR assays targeting mitochondrial and ribosomal Aspergillus DNA improves the sensitivity of PCR in the diagnosis of IA in hematological patients with risk factors and positive GM results. This study also confirms that a positive PCR result is associated with a poor prognosis in these patients and should lead to specific antifungal therapy being introduced immediately.

Exposure to moulds and actinomycetes in Alpine farms: a nested environmental study of the PASTURE cohort.

Several studies have suggested that children exposed to a farm environment are protected against allergies and asthma. The present work is an environmental study nested within the PASTURE cohort and includes 97 farmers and 74 non-farmers in three regions of the Alpine Arc (Switzerland, France and Germany). The objectives were to determine and compare the fungi and actinomycetes present in farming and non-farming environments (children's bedrooms and cowsheds), and to identify the agricultural practices associated with an increase in airborne fungi and actinomycetes in cowsheds. Air samples were collected by air pump and were analysed by culture and by direct counting of spores on membranes. During their stay in bedrooms, children living on farms were exposed to significantly greater amounts of Absidia spp., Eurotium spp., Cladosporium spp., Penicillium spp. and mesophilic actinomycetes than children who did not live on farms. Depending on the season, the levels of moulds, yeasts and actinomycetes were from 14 to 82 times higher in cowsheds before feeding the cattle than in children's bedrooms, and from 12 to 464 times higher in cowsheds after feeding than in children's bedrooms. Feeding cattle in cowsheds was associated with a significant peak in airborne moulds and actinomycetes, and this peak was higher in winter than in summer. Silage distribution was associated with low amounts of moulds and actinomycetes. Other significant agricultural factors were the type of cowshed, cowshed volume, method of food distribution to cattle and use of fresh grass. An assessment of the microbiological diversity on farms and in children's rooms may help to determine the factors protecting children from asthma and atopic diseases.

Comparative assessment of enzyme-linked immunosorbent assay and Western blot for the diagnosis of toxocariasis in patients with skin disorders.

Background The link between various chronic skin disorders and toxocariasis was previously demonstrated by case reports and several case-control studies. However, these previous studies were based only on the Toxocara canis excretory-secretory-enzyme-linked immunosorbent assay (TES-ELISA) serological technique, which is not specific due to cross-reactivity with parasites of the genera Anisakis or Ascaris. Immunoblot analysis is highly specific and can detect very low levels of Toxocara antibodies. Therefore, this technique may be useful in the identification of Toxocara infection in patients with chronic skin disorders. Objectives Because urticaria and pruritus/prurigo are skin conditions previously associated with toxocariasis, we carried out a prospective study using both TES-ELISA and Toxocara Western blot on 113 patients with either chronic urticaria (n = 84) or chronic pruritus (n = 29). Methods Patients were matched with controls according to gender, age and residence location (rural or urban area). Data were analysed using a Mantel-Haenszel chi(2) test. Results The proportion of positive TES-ELISA results was not significantly different for patients with chronic skin disorders (urticaria or pruritus/prurigo) from that of control subjects. However, the proportion of positive immunoblot results was significantly higher for patients with chronic urticaria than for control subjects (P = 0.009). Conclusions Our study demonstrates the need to perform Western blotting immunodiagnosis, whatever the TES-ELISA result, to improve diagnosis of human toxocariasis in patients with chronic urticaria caused by Toxocara infection.

False-positive Aspergillus real-time PCR assay due to a nutritional supplement in a bone marrow transplant recipient with GVH disease.

PCR screening for circulating DNA, especially when combined with antigen testing, has shown promise for the definitive diagnosis of invasive aspergillosis. False positives for Aspergillus real-time PCR assays have been described in several reports, but no sources of fungal DNA contamination could be clearly identified. We report a false-positive case for both galactomannan (GM) antigenemia and Aspergillus PCR due to nutritional supplement intake in a bone marrow transplant recipient with digestive graft-versus-host disease. Our case report also suggests that fungal DNA can pass into the serum from the intestinal tract in the same way as fungal GM. Clinicians should be aware of this possibility, so that the administration of costly, unnecessary antifungal treatments with potential adverse side-effects can be avoided.

Azole-resistant Aspergillus fumigatus: A global phenomenon originating in the environment?

Aspergillus fumigatus is the predominant etiological agent of invasive aspergillosis (IA), a difficult-to-manage fungal disease associated with a high case fatality rate. Azole antifungals, particularly voriconazole, have significantly improved the survival rate of patients with IA. However, the clinical advances made possible through the use of medical azoles could be threatened by the emergence of azole-resistant strains which has been reported in an ever-increasing number of countries over the last 10 years. The major resistance mechanism, that combines point mutation(s) in the coding sequence of cyp51A gene and an insertion of a tandem repeat in the promoter region of this gene which leads to its overexpression (TR34/L98H and TR46/Y121F/T289A), is presumed to be of environmental origin. However, the emergence of clinical and environmental azole-resistant strains without the cyp51A gene mutation suggests that other mechanisms could also be responsible for azole resistance (for example, overexpression of efflux pumps). The development of resistance may be linked to either long-term use of azole antifungals in patients with chronic aspergillosis (patient-acquired route) or selection pressure of the fungicides in the environment (environmental route). The fungicide-driven route could be responsible for resistance in azole-naive patients with IA. This literature review aims to summarize recent findings, focusing on the current situation of azole-resistance in A. fumigatus, and provides better understanding of the importance of the environmental route in resistance acquisition.

Indoor mold concentration in Eastern France.

UNLABELLED: Our prospective case-control study of 118 dwellings in Eastern France examined fungal contamination in unhealthy dwellings (n = 32) (homes with visible mold contamination and adverse health outcomes reported by the occupants), dwellings occupied by allergic patients (with medical diagnostic and positive prick-tests for molds) (n = 27) and matched control dwellings (n = 59). Unhealthy dwellings present higher airborne concentrations of Aspergillus, Penicillium, and Cladosporium than control dwellings, irrespective of the room sampled. Bedroom walls were more highly contaminated by molds than others. Dwellings occupied by allergic patients differed significantly for airborne concentrations of Penicillium only, but not for wall surface contamination, whereas bathroom walls were more highly contaminated than other rooms. Molecular identification of 12 Penicillium species showed Penicillium chrysogenum and Penicillium olsonii to be the two main species. From the total average of molds, by impaction method, useful thresholds can be given: below 170 CFU/m(3), between 170 and 560 CFU/m(3), 560 and 1000 CFU/m(3) and above 1000 CFU/m(3), respectively for dwellings with low, moderate, high, and very high concentrations. The latter would be considered a potential health hazard. PRACTICAL IMPLICATIONS: A single measure of airborne concentrations of molds by impaction allows to establish useful thresholds by social services to estimate in a objective way the housing moldiness. Excluding the summer period, reproducibility of this kind of measure on 3 months, in the fixed limits, is 94.3%. The differences in terms of biodiversity of the unhealthy housing and those accommodating allergic patients imply a specific approach to decrease fungi airborne concentrations. The biodiversity of Penicillium raises the problem of the use of the single extract of Penicillium chrysogenum for skin-tests. The extent of the contaminated surfaces must be measured to assess the potential risk linked to spore contamination. Indeed, surface sampling mostly allows qualitative assessment of the environment.