Neutrophilic eccrine hidradenitis in two patients treated with BRAF inhibitors: a new cutaneous adverse event.

Neutrophilic eccrine hidradenitis (NEH) is a rare neutrophilic dermatosis, first described in patients undergoing chemotherapy for a malignant haemopathy. It has polymorphous clinical features and the association of both clinical and histological features is necessary to make a diagnosis. We report the first two cases of NEH in patients treated with a BRAF inhibitor (BRAFi), either dabrafenib or vemurafenib, for a stage IV metastatic melanoma. Disseminated erythematous plaques associated with fever and polyarthralgia occurred early after the initiation of treatment and were badly tolerated. Histological analyses confirmed the diagnosis of NEH. Symptoms disappeared a few days after the cessation of treatment and introduction of topical steroids. The replacement of one BRAFi with another is a therapeutic alternative as it is not necessarily associated with a relapse of NEH. NEH can be added to the spectrum of neutrophilic dermatoses induced by BRAFis. It occurs earlier (3-4 days) than previously described drug-induced NEH (9-12 days) and may be an earlier stage of eccrine squamous syringometaplasia, which has already been reported in the context of BRAFi-treated patients.

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.

Evaluation in usual practice of the bevacizumab-FOLFIRI combination for the first-line treatment of patients with unresectable metastatic colorectal cancer treated in 2006: focus on resected patients and oncogeriatrics: AVASTIN OUEST cohort of the Observatory of Cancer of the Brittany and Pays de la Loire Areas (Observatoire dédié au Cancer Bretagne / Pays de la Loire).

BACKGROUND: In 2006, bevacizumab, a targeted therapy agent was combined with FOLFIRI for the firstline treatment of patients with unresectable metastatic colorectal cancer. METHODS/RESULTS: A study on a homogenous series of 111 patients from the Brittany and Pays de la Loire areas who received bevacizumab-FOLFIRI as first-line treatment in 2006 showed the following results: 51 responses, 29 stabilisations, 21 progressions and 10 cases of toxicity prior to assessment. Median overall survival (OS) was 25.1 months and median progression-free survival was 10.2 months. Surgery secondary to treatment tripled median OS which reached 59.2 months in resected patients versus 18.8 months in unresected patients. Comparison of patients aged more or less than 70 years showed no differences in terms of benefits or risks. CONCLUSION: Bevacizumab-FOLFIRI could be administered as part of a routine care protocol to elderly patients previously evaluated by a geriatric assessment and validated by a multidisciplinary staff.

En 2006, bevacizumab-FOLFIRI représente la thérapie ciblée administrable dès la première ligne chez les patients porteurs d'un cancer colorectal métastatique non opérable. Une série homogène de 111 patients colligés en région Bretagne et Pays de la Loire ayant reçu du bevacizumab- FOLFIRI en première ligne en 2006 révèle les résultats suivants: 51 réponses, 29 stabilités, 21 progressions et 10 toxicités avant évaluation. La médiane de survie globale (OS) est de 25,1 mois et la médiane de survie sans progression (PFS) de 10,2 mois. Dans le cas d'une chirurgie secondaire, l'OS médian triple de 18,8 mois chez les patients non réséqués versus 59,2 mois ceux réséqués. En comparant les sujets âgés de plus et de moins de 70 ans, aucune différence n'a été mise en évidence en termes de bénéfice ou de risque. Bevacizumab-FOLFIRI pourrait être administré en pratique courante chez les personnes âgées sous couvert d'une évaluation gériatrique et d'une approche multidisciplinaire.

Indoor dust and air concentrations of endotoxin in urban and rural environments.

SIGNIFICANCE AND IMPACT OF THE STUDY: Rural dairy farming is associated with high exposure to indoor endotoxins as compared to rural nonfarming houses and urban houses. The time spent on the mattress (7 h for an adult) and of the proximity of the contaminated source should be taken into account with the other causes of exposure. Studies in European children from a farming background have shown that these children have a reduced risk of asthma and atopic sensitization compared to their urban counterparts. It has been suggested that this might be due to exposure to high levels of endotoxin in the farming environment. The aim of this study was to compare indoor endotoxin concentrations in air and dust samples from randomly selected urban and rural dwellings. In the rural area, endotoxins were analysed in farmhouses and nonfarmhouses as well as housing characteristics, lifestyle factors and agricultural practices likely to influence air and dust endotoxin levels. Endotoxin levels were significantly higher in floor (6600 ± 6100 vs 3600 ± 5600 and 3800 ± 17,000 ng g⁻¹; P < 0·001) and mattress dust (2900 ± 4100 vs 1100 ± 2400 and 800 ± 2600 ng g⁻¹; P < 0·001) from farmhouses compared to other rural and urban homes. However, no difference was observed between endotoxin concentrations in the air of urban and rural houses, and airborne endotoxin levels did not correlate to dust levels. Lack of ventilation and direct entry into the house were correlated with an increase in dust endotoxin levels. These results confirm that dairy farming is associated with high exposure to endotoxins in indoor dust samples. No difference was observed between indoor airborne concentrations between urban and rural houses. These results suggest that measuring endotoxin in dust is the most relevant method to assess endotoxin exposure.

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.

Impact of an intensive multimodal educative program on behavioral disorders of polyhandicapped patients: A randomized controlled trial.

BACKGROUND: Hospitalized children and young adults with polyhandicap (PLH) often present with behavioral and relational disorders that are mainly related to their difficulties in communicating and interacting with their environments. Educational support is rarely provided to these patients. An intensive multimodal educative program could help in reducing behavioral disorders and in improving the quality of life of healthcare workers, including nurses and auxiliary nurses. METHODS: A multicenter, randomized controlled trial compared the impact of the usual practice of an educative program (1 h a week) to a multimodal intensive educative program (5 h a week) at 12 months. Patients aged 3-25 with PLH defined by the combination of five criteria (motor deficiency, severe-to-profound mental impairment, daily life dependence, restricted mobility, onset of cerebral lesion at younger than 3 years, and at least one behavioral disorder per week [withdrawn behavior, unexplained crying, teeth grinding, self-injury, aggression, stereotypy, or merycism]) were included. The primary outcome was the evolution of the predominant behavioral disorder between study inclusion and 12 months. Healthcare workers completed questionnaires about chronic stress, coping strategies, and quality of life at study inclusion and at 12 months. RESULTS: Overall, 60 patients were included. Despite a tendency toward reduced teeth grinding, withdrawn and self-injury behaviors, the intervention was not significantly effective: The median duration of continuous behavioral disorders (stereotypy, unexplained crying, withdrawn behavior, and teeth grinding) did not differ between groups. The median frequency of the discontinuous behavioral disorders (self-injury) did not differ between groups. Considering each disorder separately, there was a decrease in teeth grinding, self-injury, and autistic-like traits in the intervention group, although it did not reach statistical significance. This study also suggested decreased depersonalization feelings by healthcare workers. CONCLUSION: Although the study did not show a significant reduction in behavioral disorders in patients with PLH, these results encourage further evaluation of educational management, particularly in regard to patients with self-injury and with withdrawn and teeth-grinding behaviors.

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.

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.

Neurogenic inflammation in the upper digestive tract of the mule duck: effect of a chemical algogen and force-feeding.

1.The objectives were to quantify the presence of neurogenic inflammation in 4 regions of the upper digestive tract of anaesthetised ducks (post-pharynx, pseudo-crop, transition between the pseudo-crop and the proventriculus, and proventriculus) after application of HCl stimulation of up to 4 M in the pseudo-crop. 2.The second objective was to quantify the presence of neurogenic inflammation in the same digestive tract regions as mentioned above during 4 feeding periods of foie gras production (rearing, preparation to force-feeding, and second and last meals of the force-feeding period). 3. Extravasation increased above a HCl stimulation threshold of 2 M. Furthermore, more extravasation was observed in the proventriculus compared to the other regions (P < 0·001). 4.Highest extravasation responses were observed in the proventriculus and the pseudo-crop at the end of the preparation period, and in the proventriculus after the second forced meal, compared with the rearing period (P < 0·01), with a return to rearing level at the end of force-feeding. 5.Such a kinetic could be indicative of a relative mildness of the irritant components associated with this feeding practice.

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.

Medium-term effects of repeated exposure to stray voltage on activity, stress physiology, and milk production and composition in dairy cows.

The medium-term effects of permanent or random exposure to stray voltage applied to the water trough were evaluated on milk production and stress physiology in lactating dairy cows. Seventy-four Holstein cows were assigned during two 8-wk experimental periods to 1 of 3 treatments. The treatments were permanent exposure to voltage (PERM, 1.8 V, n=23) applied to the water trough, random exposure to voltage (RAND, 1.8 V, 36 h/wk, n=25), and no exposure to voltage (control, n=26). On the first day of voltage exposure, PERM cows had higher activity levels than control cows (9.8+/-2.70 vs. -2.3+/-2.74 14-s periods of movement/h). During the eighth week of exposure, RAND cows had higher activity levels than control cows (4.2+/-3.64 vs. -7.7+/-3.54 14-s periods of movement/h) and higher milk cortisol concentration than PERM cows (0.21+/-0.024 vs. 0.14+/-0.020 ng/mL). No differences were observed between treatments for cortisol response after an ACTH challenge during the seventh week of exposure. No effects of voltage exposure were observed on production traits and daily water intake. There was a transient decrease in milk yield on the second day of exposure in PERM cows (-1.4+/-0.74 kg) and on the third day of exposure in RAND cows (-3.5+/-1.03 kg) compared with control cows. In dairy cows, permanent or random exposure to stray voltage (1.8 V; 3.6 mA) could induce a transient stress response. Moreover, unpredictable voltage exposure could be considered a mild stressor, with slight modifications in stress physiology and activity but no impairment in production in the medium term.

[Treatment of the lower urinary tract symptoms secondary to benign prostatic hyperplasia by phosphodiesterase type 5 inhibitors. Review article]. / Traitement des troubles du bas appareil urinaire liés à une hyperplasie bénigne de prostate par inhibiteur de la phosphodiestérase de type 5.

CONTEXT: In aging males, there is an increased prevalence of lower urinary tract symptoms (LUTS) related to benign prostatic hyperplasia (BPH) and erectile dysfunction (ED) whose affect significantly their quality of life. LUTS would represent for ED an independent risk factor. Some treatment of LUTS have undesirable effects on the erectile function. The phosphodiesterase type 5 inhibitors (IPDE 5) revolutionized the treatment of ED. Several recent clinical studies evaluated the effect daily treatment by IPDE 5 on the LUTS secondary to BPH among patients with or without ED. MATERIALS AND METHODS: we searched Medline for the peer-reviewed articles in English published, pertaining to findings of potential interest supporting a role of IPDE5 in LUTS due to BHP. The keywords used were: benign prostatic hyperplasia; cyclic nucleotide phosphodiesterase type 5; lower urinary tract symptoms; erectile dysfunction. RESULTS: Generally, daily treatment with IPDE 5 improves the LUTS secondary to BPH as well as in both the storage and voiding domains of the international prostate symptom score (IPPS) and in quality of life questionnaire. It was not observed adverse events. CONCLUSION: The first results of the use of IPDE 5 in the LUTS treatment secondary to the HBP seem promising. However, a direct comparison of efficacy of IPDE 5 and alpha-blockers or 5-alpha-reductase inhibitors is not yet available. The mechanism(s) of action(s) of these compounds in this indication remain difficult to affirm even if new hypotheses can be formulated.

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.

Indoor fungal contamination of moisture-damaged and allergic patient housing analysed using real-time PCR.

AIMS: The aim of our study was to compare, using real-time (Rt) PCR, quantitative levels of five fungal species in three kinds of dwellings. METHODS AND RESULTS: Three groups of homes were recruited: moisture-damaged homes (MDH, n = 30), allergic patient homes (APH, n = 25) and paired control homes (CH, n = 55). Five moulds with allergenic compounds or mycotoxin production characteristics (Cladosporium sphaerospermum, Penicillium chrysogenum, Aspergillus versicolor, Alternaria alternata and Stachybotrys chartarum) were quantified using Rt-PCR. Cycle threshold results were expressed in spore equivalent per volume or surface unit using a direct calculation based on a spore standard curve. MDH presented significantly higher amounts of DNA from C. sphaerospermum in both air and surface samples than CH (P < 0.001). APH presented slightly elevated amounts of DNA from A. versicolor in both air and surface samples, compared to CH (P < 0.05). CONCLUSION: Rt-PCR quantification of targeted fungal species is a rapid, reliable tool that could be included in a global indoor mould evaluation. SIGNIFICANCE AND IMPACT OF THE STUDY: Quantification of C. sphaerospermum using Rt-PCR can help to better target social service intervention in MDH. Quantification of A. versicolor DNA could be informative for characterization of APH.

Excess of infections due to a multi-drug sensitive Salmonella enterica serotype Typhimurium in France in June 2008.

An unusually high number of cases of Salmonella Typhimurium was reported in France in June 2008. In the course of epidemiological investigations 112 cases were ascertained, of whom 75 were interviewed. Subtyping by PFGE and MLVA identified a strain named "majority profile". Subtyping results were available for 45 interviewed cases, 30 of whom (majority below 15 years of age) were found to be infected with the majority profile strain. Evidence suggested the occurrence of an outbreak due to a monoclonal S. Typhimurium strain with the single PFGE profile XTYM-50. Cases with identical PFGE profile were also detected in Switzerland but no link with outbreaks occurring in the same period in Denmark and in the Netherlands was found. Contamination of a product distributed nationally was suggested as the cause of the outbreak but investigations did not reveal any specific food source.

Comparison of different techniques for inoculation of "Candidatus Phytoplasma mali" on apple and periwinkle in biological indexing procedure.

As phytoplasmas are non cultivable micro-organisms, the research on phytoplasmal diseases can only be achieved with infected hosts. Biological indexing (by grafting) is the simplest detection method for phytoplasmal diseases. We tested four different grafting techniques for inoculation of apple trees or periwinkles in greenhouse, including whip graft, bark graft, budding and chip-budding. All techniques were tested on apple trees (six trees per phytoplasma isolates) in insect-proof greenhouse. The whip and bark grafting were not feasible for periwinkle plants, because of fineness and fragility of their tissues: only the chip-budding was performed (four plants per isolate). In apple trees, the best and soonest positive results were obtained by chip and bark grafting. Except for seven transplants not-grown after grafting, 100% efficiency of inoculation was obtained by both methods. Nevertheless, the transmission of phytoplasma from transplant not-grown to rootstock was sometimes recorded (28.6%). The earliest phytoplasma symptoms after whip or bark grafting appeared after 3 months. Symptoms were obtained much later with budding and chip-budding. In case of periwinkles, infected apple and periwinkle materials were used as inoculum sources. Transmission of phytoplasma from periwinkle to periwinkle was successfully carried out by chip-budding grafting. The symptoms were observed during the second month after inoculation. The transmission of phytoplasma from infected apple material to periwinkle (by chip-budding) was achieved for 60 % of the tested samples. Moreover, the latency period before symptom observation was longer. Finally, we perceived the apple trees are more convenient and rapid than periwinkle plants for biological indexing of apple materials.

[Classical and molecular methods for identification and quantification of domestic moulds]. / Méthodes d'identification et de quantification des moisissures de l'habitat : méthodes classiques, méthodes moléculaires.

INTRODUCTION: To study the impact of the constant and inevitable inhalation of moulds, it is necessary to sample, identify and count the spores. BACKGROUND: Environmental sampling methods can be separated into three categories: surface sampling is easy to perform but non quantitative, air sampling is easy to calibrate but provides time limited information, and dust sampling which is more representative of long term exposure to moulds. The sampling strategy depends on the objectives (evaluation of the risk of exposure for individuals; quantification of the household contamination; evaluation of the efficacy of remediation). The mould colonies obtained in culture are identified using microscopy, Maldi-TOF, and/or DNA sequencing. VIEWPOINTS: Electrostatic dust collectors are an alternative to older methods for identifying and quantifying household mould spores. They are easy to use and relatively cheap. Colony counting should be progressively replaced by quantitative real-time PCR, which is already validated, while waiting for more standardised high throughput sequencing methods for assessment of mould contamination without technical bias. CONCLUSION: Despite some technical recommendations for obtaining reliable and comparable results, the huge diversity of environmental moulds, the variable quantity of spores inhaled and the association with other allergens (mites, plants) make the evaluation of their impact on human health difficult. Hence there is a need for reliable and generally applicable quantitative methods.

Establishment of a new method for rapid and precise estimation of apple proliferation phytoplasma concentration in periwinkle.

Quantification of a plant pathogen is essential to study its population dynamic in various conditions and to relate symptom expression with pathogen concentration. Up to now, very few methods have been published to quantify phytoplasmas. So, the objective of this work was to establish a method able to quantify the Apple Proliferation (AP) phytoplasma populations in periwinkles. The present work was based on a method previously published to detect AP phytoplasma. This method was optimized to transform it into a quantitative method. First, a new probe specific for AP detection was applied. This probe successfully detected only AP isolates (versus closely related ESFY and PD phytoplasmas). Secondly, the method was adapted to allow the quantification of phytoplasma in periwinkle leaves. For quantification, the calibration curve was built on serial dilutions of a plasmid containing the amplified fragment (phytoplasma 16Sr gene). The limit of detection of the method was one copy of cloned phytoplasma DNA in the reaction while the lower and upper limits of quantification were 102 and 108. Sample DNA extracts were diluted 100X before amplification and standards were prepared in 100x diluted DNA extract from healthy plant. Using the calibration curve, the concentrations in the tested samples were calculated at 2 x 10(5) to 10(6) individuals per mg of fresh midrib. This work is a preliminary step to study the interaction of phytoplasmas with their hosts in relation to symptoms expression.