A molecular approach for the rapid, selective and sensitive detection of Exophiala jeanselmei in environmental samples: development and performance assessment of a real-time PCR assay.

Exophiala jeanselmei is an opportunistic pathogenic black yeast growing in humid environments such as water reservoirs of air-conditioning systems. Because this fungal contaminant could be vaporized into the air and subsequently cause health problems, its monitoring is recommended. Currently, this monitoring is based on culture and microscopic identification which are complex, sometimes ambiguous and time-demanding, i.e., up to 21 days. Therefore, molecular, culture-independent methods could be more advantageous for the monitoring of E. jeanselmei. In this study, we developed a SYBR®green real-time PCR assay based on the internal transcribed spacer 2 from the 18S ribosomal DNA complex for the specific detection of E. jeanselmei. The selectivity (100 %), PCR efficiency (95.5 %), dynamic range and repeatability of this qPCR assay were subsequently evaluated. The limit of detection for this qPCR assay was determined to be 1 copy of genomic DNA of E. jeanselmei. Finally, water samples collected from cooling reservoirs were analyzed using this qPCR assay to deliver a proof of concept for the molecular detection of E. jeanselmei in environmental samples. The results obtained by molecular analysis were compared with those of classical methods (i.e., culture and microscopic identification) used in routine analysis and were 100 % matching. This comparison demonstrated that this SYBR®green qPCR assay can be used as a molecular alternative for monitoring and routine investigation of samples contaminated by E. jeanselmei, while eliminating the need for culturing and thereby considerably decreasing the required analysis time to 2 days.

Development and performance assessment of a qualitative SYBR® green real-time PCR assay for the detection of Aspergillus versicolor in indoor air.

Currently, contamination of indoor environment by fungi and molds is considered as a public health problem. The monitoring of indoor airborne fungal contamination is a common tool to help understanding the link between fungi in houses and respiratory problems. Classical analytical monitoring methods, based on cultivation and microscopic identification, depend on the growth of the fungi. Consequently, they are biased by difficulties to grow some species on certain culture media and under certain conditions or by noncultivable or dead fungi that can consequently not be identified. However, they could have an impact on human health as they might be allergenic. Since molecular methods do not require a culture step, they seem an excellent alternative for the monitoring of indoor fungal contaminations. As a case study, we developed a SYBR® green real-time PCR-based assay for the specific detection and identification of Aspergillus versicolor, which is frequently observed in indoor environment and known to be allergenic. The developed primers amplify a short region of the internal transcribed spacer 1 from the 18S ribosomal DNA complex. Subsequently, the performance of this quantitative polymerase chain reaction (qPCR) method was assessed using specific criteria, including an evaluation of the selectivity, PCR efficiency, dynamic range, and repeatability. The limit of detection was determined to be 1 or 2 copies of genomic DNA of A. versicolor. In order to demonstrate that this SYBR® green qPCR assay is a valuable alternative for monitoring indoor fungal contamination with A. versicolor, environmental samples collected in contaminated houses were analyzed and the results were compared to the ones obtained with the traditional methods.

The role of indoor pollution in the development and maintenance of chronic airway inflammation in children.

Air pollution is one of the great problems of this century and it plays an important role in the increasing prevalence of chronic inflammatory problems in the upper airway in children. Since their lungs and immune system are not fully developed when exposure begins, newborns and children appear to be more sensitive to the effects of both outdoor and indoor air pollution. Furthermore, children spend most of their time indoors and are exposed more often to pollutants in indoor air. The link between health problems, chemical products and allergens (the latter mainly from cats and mites) has been extensively studied. Other important indoor contaminants are fungi, which are often present in damp buildings and can cause severe respiratory disease by producing spores, allergens, volatile irritant compounds and toxins. A proper identification of mould contamination of this kind is vital for correct diagnosis, treatment and the prevention of health problems, and improvements have been observed after the removal or cleaning of the contaminated materials and improvements to the ventilation of buildings. While a possible association between respiratory symptoms, such as rhinitis, and the presence of fungi in the indoor environment has been documented by several authors, other studies have observed no significant relationship. The development of standardised sampling, detection and diagnostic tests will be essential to understand the proper role of fungi in the indoor atmosphere and their impact on public health.

The anatomical distribution of radiological abnormalities in Kashin-Beck disease in Tibet.

A radiological study of osteoarticular changes in Kashin-Beck Disease (KBD) was undertaken on the appendicular skeleton in 105 patients with KBD, in 31 healthy subjects living in an endemic area and in 30 healthy subjects living in a non-endemic area. The bone age was delayed in all three populations with no significant difference between the three studied Tibetan populations. Radiological changes occur in 56% of patients with KBD, and are usually bilateral. An analysis of the distribution of lesions shows a proximo-distal gradient. The changes are more common in the distal aspect of the limb and the lower limb is involved more commonly than the upper limb. The foot and ankle are involved in 89.5% of cases. The radiological changes and their distribution might be explained by the hypothesis of inhibition of angiogenesis by mycotoxins, exacerbated by chemical and physical environmental factors.

A 4-year study of the mycological aspects of Kashin-Beck disease in Tibet.

In order to clarify the association between mycotoxin-producing fungi in food and Kashin-Beck disease (KBD), we examined the occurrence and contamination levels of fungi in samples of barley grain, from KBD-affected families and from unaffected families in endemic areas. A control area without the occurrence of KBD served as reference. The first results obtained in 1995 showed that total mesophilic fungal contamination of barley grain was consistently higher in families with KBD. Trichothecium roseum (Pers) Link ex gray, Dreschlera Ito and Alternaria Nees ex Fr. were the three most common fungi significantly associated with KBD. In 1996 we again observed a significant difference between affected and non-affected families, especially with Trichothecium roseum and Ulocladium Preuss. On this basis, measures to prevent KBD were suggested and a preventive program has been set up since 1998 in 20 new villages.

The prevalence of mycotoxins in Kashin-Beck disease.

Mycotoxins are naturally occurring toxic chemical compounds produced by fungi infesting agricultural crops both during their growth and storage. Such secondary metabolites, when ingested, can produce toxic syndromes in humans. As it has been suggested that mycotoxins might be involved in the development of Kashin-Beck disease (KBD), we undertook a survey of barley grains of KBD-affected families and non-affected families in that country. We found, by thin layer chromatography, a hitherto unknown metabolite of Alternaria sp. This was especially common on the barley grains of KBD-affected families.

Epidemiological support for a multifactorial aetiology of Kashin-Beck disease in Tibet.

We carried out a cross-sectional study in 12 rural villages in order to identify the risk factors for Kashin-Beck disease in Tibet. Children aged 5-15 years (n=575) were examined and their corresponding houses were visited. Samples were collected in order to study fungal contamination of stored grain and the organic matter content of drinking water. Multivariate analysis was performed using logistic regression and population attributable fractions were computed to estimate the impact of each factor. The following variables were independently associated with the disease: age, gender, low socio-economic status, indicators of a poorly diversified diet, iodine deficiency and small water container size (with higher organic matter levels in small containers). Selenium deficiency was severe in all study subjects. The degree of fungal contamination of barley grain was related to the highest percentage of cases (65%) in a sample of the study population. Higher urinary iodine levels were not associated with decreasing prevalence rates when Alternaria sp. was isolated. The data that we report supports the hypothesis that Kashin-Beck disease occurs as a consequence of oxidative damage to cartilage and bone cells when associated with decreased antioxidant defence. Another mechanism that may coexist is bone remodelling stimulated by thyroid hormones whose actions can be blocked by certain mycotoxins.

[Mold allergy: 25 years of indoor and outdoor studies in Belgium]. / Allergie aux moisissures: 25 années d'études "indoor" et "outdoor" en Belgique.

Collecting atmospheric fungal spores in Brussels for 25 years revealed significant quantitative differences at three different levels: the total number of spores collected every year, the annual frequency rate of some species e.g. Cladosporium and Alternaria, the proliferation of some rare species according to weather conditions.