Abundance of domestic mites in dwellings of children and adolescents with asthma in relation to environmental factors and allergy symptoms

Exposure to house dust allergens, mainly from domestic mites, is an important cause of allergic reactions in sensitized asthmatic patients. A total of 63 dust samples were collected from 16 flats in Bytom (south Poland); in each flat a person (age 4–17 years) suffering from bronchial asthma lived with his/her family. Mite density was calculated as the number of specimens per g of dust. The results were compared with household features and the data were statistically analyzed. In total 566 mite specimens were isolated, including 526 members of the family Pyroglyphidae (93%). The dominant species were Dermatophagoides pteronyssinus (60% of the total count) and Dermatophagoides farinae (32%). Pyroglyphids were found in all mite positive samples (68%) of which 35% also contained non-pyroglyphids, including glycyphagids, cheyletids and gamasids. The results suggest associations between the density of some mite taxa (per g of dust) and the following indoor environmental factors: presence of pets, number of inhabitants, coal-stoves as a type of heating, cleaning frequency, higher relative humidity, presence of flowers and PVC windows. The severity of asthma seems to be associated with the numbers of D. farinae, total domestic mites and live mites per g of dust.


Materials and methods
The study was performed in 16 flats occupied by 16 persons suffering from bronchial asthma, with ages ranging from 4 to 17 years. All subjects were selected from the patients of Department of Pediatrics in Bytom (Medical University of Silesia in Katowice), which have agreed to participate in the study. All these patients were previously diagnosed with an allergy to dust mites. According to GINA 2006 classification, 1 person was diagnosed with severe asthma, 9 with moderate, and 6 with mild asthma. Moreover, 4 persons suffered from atopic eczema and 9 from perennial allergic rhinitis. Allergy to Dermatophagoides spp. was confirmed according to the current standards 27,28 . The results of skin prick tests with allergens Der p 1 and Der p 2 were positive, and the following levels of IgE were detected: against Der p 1-very high (> 17.5 IU/ml) in 12/16 patients, high (> 3.5-17.5 IU/ml) in 2/16 patients, moderately high (> 0.7-3.5 IU/ml) in 2/16 patients and against Der p 2-very high in 12/16 patients, in 12/16 patients-high, in 2/16-moderately high, and in 1/16-low (> 0.35-0.7 IU/ml).
The examined dwellings were located within the same urban or suburban area of the old and big city of Bytom in Upper Silesia, south-eastern Poland, and were investigated regarding the content of the house dust mites and different household environmental factors. A total of 63 dust samples were collected from the flats and examined for the presence of allergenic mites. The samples were collected from beds, upholstery, wooden furniture and floors, carpets, linoleum, as well as from other places, such as bedclothes and walls. A surface area of 1 m 2 at each sampling site was hoovered for 2 min. Next, the samples were weighed and analysed for mites, as previously described by Solarz 5 .
All mites were mounted in Hoyer's medium on slides, and the species and life stages were determined with the aid of a compound microscope. Mite density was calculated as the number of specimens per 1 g of dust.
Mites that were alive at the time of sampling could easily be distinguished as intact (undamaged) and because of their plump and/or white appearance. Next, results of these studies were compared with household data of the persons suffering from asthma. Information on various parameters which could influence the numbers of mites was obtained by questioning the residents. These parameters (explanatory variables x) were: age of the building, locality (urban or suburban), type of building (close/compact or freestanding), type of building construction (housing block, concrete or brick), level of dwelling (first floor or higher), area occupied by each flat, size of family, type of window frame (PVC or wood), type of heating (central, electric, gas, oil or coal-stove), type of floor (carpet, plastic or wood), level of humidity, presence or absence of domestic flowers/plants, presence or absence of pets, place and time (month) of sampling, and weight of samples. The following factors characterising the sleeping accommodation of the examined asthmatic patients were included in the analysis: type of sleeping accommodation (bed/mattress or couch), type of bedclothes (feather beds, quilts, blankets and pillows), and temperature in the bedroom. Additional parameters analysed were frequency of cleaning and housewife (employed or not). The criterion variables (y) on the other hand were: number of mites per gram of dust (total or live), in relation to particular pyroglyphid mite species, total house dust mites (Pyroglyphidae), total domestic mites (including non-pyroglyphid allergenic mites-acarids, glycyphagids), and total mites (including other taxa). The data obtained was analysed using the Kolmogorov-Smirnov test, the Spearman's rank correlation test, Mann-Whitney U test, Wilcoxon signed-rank test and χ 2 test.

Results
Overall results. The age of buildings varied from 10 to 105 years ( x = 51.61). The weight of samples ranged from 0.01 to 2.4 g ( x = 0.24). The results obtained are presented in Tables 1, 2 (Table 2). Among 25 samples with a single mite species, 18 samples contained D. farinae and only 7 of them D. pteronyssinus. 9 samples were co-inhabited by two species of mites, 7 types of the species composition were noted, and most frequently the combination of D. pteronyssinus and a member of the family Cheyletidae (3 samples) occurred. All of these combinations of mite species composition, which were observed in dust samples from dwellings, are listed in Table 2. This comparison also includes mites of other taxa. No mites were found in 20 samples (31.7%). The majority of domestic mite species occurred with low frequency (Table 1). Therefore, for these species and for some dwellings, the median values of numbers of mites per gram of dust were 0.0. D. farinae was the most frequent species and it was found in 29 samples (46.03% of the total count and 67.44% of samples positive for mites) (Table 1), whereas D. pteronyssinus was found in 21 samples (33.3% of the total count). The difference was statistically non-significant (Yates corrected χ 2 = 3.01, p = 0.083).  Table 1. The numbers were approximately 5.4 and 2.9 for D. pteronyssinus and D. farinae, respectively. Densities (numbers of mites per gram of dust) of the total mite population, total domestic mites, total pyroglyphids or particular pyroglyphid mite species varied from one dwelling to another, and from one location to another within the dwellings. In bedrooms, the allergenic mites, both house dust mites and total domestic mites, were most abundant on pillows and on quilts, whereas they were much less numerous on sheets. Generally, D. farinae was more abundant in bed dust samples than D. pteronyssinus.
Relations between the household characteristics, bronchial asthma and domestic mites. The highest number of mite species was found in a 105 year old house, 3 species in 70-year old, 50-year old and 10-year old houses. The persons who lived in these houses showed the symptoms of moderate, episodic, severe and moderate asthma respectively. The mean age of the buildings where the asthmatic patients with moderate asthma lived was 59 years, and with episodic asthma 58 years. Most patients suffering from episodic asthma (5/6 persons; 83.0%) lived in detached houses on the city outskirts, built of concrete slabs or bricks. Most patients suffering from moderate asthma (6/9 persons, 67.0%) lived in high-density housing in the center of Bytom (tenement houses). No D. pteronyssinus was found in the samples from 3/4 of the flats located in the buildings with concrete slabs as well as in the tenement houses. Only 3/16 of the flats were located above the first floor (II, III, IX). No mites were found in the dust samples collected from the first two flats, two mite species were recorded in the third flat, however the number of mites in 1 g of dust was fairly small (max. 12.9). Only two flats were heated by coal stoves; D. pteronyssinus and D. farinae were present in both flats and the number of mites in 1 g of dust was high (max. 933.4 and 380.0). The samples of dust taken from 4 flats with insulated windows with plastic frames showed various numbers of mites; in two flats they were high (max. 466.7 and 5000/1 g), and in two low (max. 24.1 and 80.0/1 g). Additionally, the type of floors and signs of dampness in the flats did not show any connections with the species and concentration of mites in the dust.
The majority of the data actually obtained was not distributed normally (Kolmogorov-Smirnov test; p < 0.01 or p < 0.05).
The number of pyroglyphid mites per gram of dust (both total and live mites) in the examined dwellings was significantly associated (Wilcoxon signed-rank test) with the number of inhabitants (family size) (p < 0.001), coal-stoves as type of heating (p < 0.001), higher cleaning frequency (p = 0.000004), dampness (p < 0.001), presence of flowers (p < 0.001), employed housewife (p < 0.001) and PVC windows (p < 0.001), whereas bedroom temperature was significantly associated only with the number of live pyroglyphids per gram of dust (Wilcoxon signed-rank test, p < 0.001). Number of D. farinae per 1 g of dust was significantly associated (Wilcoxon signedrank test) with presence of pets (p < 0.001), coal-stoves as type of heating (p ≤ 0.01), dampness (p = 0.04), presence of flowers (p ≤ 0.005), employed housewife (p ≤ 0.02) and PVC windows (p ≤ 0.01).
Higher relative humidity in flats significantly influenced only the higher numbers of live D. farinae and live D. pteronyssinus per 1 g of dust (Mann-Whitney U test; p = 0.036 and 0.0074, respectively). Free standing houses had significantly higher number of D. farinae per gram of dust (Mann-Whitney U test; p = 0.0051). Typical beds (with mattresses) contained significantly higher numbers of D. farinae, live house dust mites and live domestic mites per 1 g of dust (Mann-Whitney U test; p < 0.01, p = 0.012; p < 0.05, respectively). Moreover densities of D. Table 3. Relationships observed between housing conditions and the prevalence of mites in dust samples from dwellings of the examined patients in Bytom (south Poland). Results of the Spearman's rank correlation test analysis (*p < 0.05). Part I. Type of building: block of houses [1], tenement-house [2], brick house (old) [3]; Type of heating: central [1], electric/gas/oil [2] or coal-stove [3]; Age of building: in years; Pets: no pets [0], fish/reptilians [1], birds [2], mammals (cats, dogs, hamsters, rabbits) [3]; Family size: number of inhabitants; Flat size: area occupied by each flat (surface in square meters); House wife: not employed [1], employed [2]. www.nature.com/scientificreports/ farinae (per gram of dust) were associated with suburban localisation of the house, with PVC windows and with flats located in the brick houses (Mann-Whitney U test; p < 0.05, p < 0.005 and p < 0.01, respectively). The severity of asthma was significantly associated with the numbers of D. farinae, total house dust mites, total domestic mites and live house dust mites per gram of dust (Wilcoxon signed-rank test; p = 0.0051, p < 0.001, p < 0.001 and p = 0.00012, respectively). Rhinitis was significantly associated with the total house dust mites and total domestic mites per gram of dust (Wilcoxon signed-rank test; p < 0.001, in both cases).
The results of the Spearman's rank test for correlation between some abiotic and biotic indoor environmental factors (housing conditions) and mite prevalence and density in the examined dwellings suggests associations between density of some mite taxa (per gram of dust) and the following abiotic or biotic indoor environmental factors: type and age of building (D. pteronyssinus with old brick houses, D. farinae with block houses and new houses), type of heating (D. pteronyssinus with coal-stoves), presence of pets (positive influence on densities of domestic or total mites and live D. pteronyssinus), family size and flat sizes (positive influence of smaller families or flats), suburban localisation (on D. farinae), place of sampling (beddings and beds on pyroglyphids, domestic and total mites), higher weight of sample (with exception of live D. farinae), type of sleeping accommodation (mattresses on live pyroglyphids and couches on D. farinae, total pyroglyphids, total domestic mites or total mites), absence of flowers/plants (on live D. farinae), lower level/floor of a flat (on D. farinae, total pyroglyphids, total domestic mites and total mites), type of windows (PVC on D. farinae and wooden on live pyroglyphids), higher cleaning frequency and higher moisture. Other correlations were nonsignificant (p > 0.05) ( Tables 3, 4 and 5).
Moreover, some correlations were observed between parameters of allergic diseases and the prevalence and density of mites in samples from dwellings of the examined allergic patients ( Table 6). The severity of asthma was mainly correlated with the density of D. farinae per gram of dust, as well as the atopic dermatitis and allergic rhinitis. Higher levels of specific IgE to D. pteronyssinus and D. farinae were correlated positively with higher densities of D. farinae, total pyroglyphids, total domestic mites and total mites per gram of dust, whereas sensitivity on other aeroallergens was positively correlated with the density of D. pteronyssinus. Other correlations, both positive and negative, significant or insignificant, are presented in Table 6.
Many significant relationships (positive and negative) were found between housing conditions and characteristics of allergic patients or some parameters of allergic diseases (Wilcoxon signed-rank test). Age and type of the building (old building, brick building) were significantly associated with rhinitis, atopic dermatitis, severity of asthma, higher IgE levels both for D. pteronyssinus and D. farinae, and reactivity on other allergens (other than domestic mites) (p < 0.001 in all cases). The influence of the presence of pets was also significant for rhinitis (p < 0.001), atopic dermatitis (p < 0.001), severity of asthma (p < 0.05), higher IgE levels both for D. pteronyssinus and D. farinae (p < 0.001), and reactivity on other allergens (other than domestic mites (p < 0.001).

Number of mites (per 1 g of dust)
Dwelling   www.nature.com/scientificreports/ It should also be stressed that some significant correlations (positive and negative) were found between housing conditions, characteristics of allergic patients and some parameters of allergic diseases ( Table 7). The following important significant correlations (p < 0.05) were found (among many others presented in Table 7): 1. familial predisposition to atopy was negatively correlated with the age and type of building, working housewife, type of sleeping accommodation (bed mattresses), higher humidity and higher bedroom temperature, whereas it was positively correlated with higher family size, the presence of flowers/plants, type of windows (PVC) and higher cleaning frequency; 2. severity of asthma was negatively correlated with a working housewife and type of sleeping accommodation (bed mattresses), whereas it was positively correlated with higher family size, type of windows (PVC), bedclothes (pillows), couches as the type of sleeping accommodations and a higher cleaning frequency; 3. atopic dermatitis was correlated with central heating, free standing houses, not employed house-wife, couches or sofas as types of sleeping accommodation, lower level/floor, type of windows (PVC), lower humidity, and with age (higher) and sex (male). 4. allergic rhinitis was correlated with the lower age of building, free standing buildings, absence of flowers/ plants, PVC surface of floors in the bedroom, type of windows (PVC), lower bedroom temperature, and age (higher) and sex (male); 5. sensitivity on other aeroallergens was correlated with older brick buildings, smaller flats, couches and sofas, higher cleaning frequency and a higher bedroom temperature, age (higher) and sex (male). www.nature.com/scientificreports/ Other positive and negative correlations, including levels of specific IgE and month of intensification of asthma symptoms, are presented in Table 7.

Discussion
For the first time in Poland occurrence of house dust mites was investigated in relation to types of dwellings and indoor environmental conditions, possibly affecting the development of asthma in children and adolescents with house dust mite allergy. Pyroglyphid mites usually make up 60-90% of the house dust acarofauna in temperate climate regions throughout the world 12 , also in Poland 4,7,29 . Most often they are found in habitats intimately associated with humans, such as beds, couches, sofas, other upholstery furnitures, clothing, floors and carpets 4,7,9,10,29 . Two mite species D. pteronyssinus and D. farinae are most often found and most abundant in house dust throughout the world 4,7,9,10,12,14,19,29 . Laboratory culture studies showed that dust mites do not tolerate relative humidity below 60% (or 55% for D. farinae) 1-3,5,12,30-33 . However, many house dust surveys suggest a lower limit of relative humidity for dwellings, corresponding to 45% at 20-25 °C. Below these temperatures mites will desiccate and die 1,5,12,[30][31][32][33] . In certain studies on the other hand, an indoor humidity of 45% RH corresponds to a level of mite density of 100 mites per gram of dust (the threshold value of the risk of exposure to house dust mites) and higher [1][2][3]12 . It is commonly known that dust mites are more resistant to desiccation at lower temperature values because their Critical Equilibrium Activity (CEA) (or Critical Equilibrium Humidity-CEH) is dependent on the air temperature level, and stays lower with its decrease 1 . Dust mite populations are also found worldwide in dwellings where indoor relative humidity periodically fluctuates on much lower levels than 65% for extended periods of time during each 24 h 1,5,12,26 . At relative humidities ranging between 30-50%, D. farinae mites are less susceptible to desiccation than D. pteronyssinus, which was almost completely absent in the examined samples 5,12 . Ratios of numbers of the particular pyroglyphid dust mite species, especially between D. pteronyssinus and D. farinae, differ in various regions of the world. Decisive factors influencing their occurrence and abundance are mainly relative humidity and temperature of both outdoor and indoor air [1][2][3][4]7,12,34 . It is commonly known that the optimal temperature is higher (25-30 °C) and the optimal humidity is lower (50-75% RH) for D. farinae than D. pteronyssinus. The first species appears to survive better in dryer habitats than the latter. Lower temperature (15-20 °C) and higher humidity (75-80% RH) favour the survival of D. pteronyssinus in mixed laboratory cultures 4,9,10,12,16 . Domination of D. pteronyssinus in dwellings appears to be a common tendency at many localities in Poland 7,27 . This tendency may also indicate that dwellings at these localities in our country are more humid. On the other hand, the indoor air ambient humidity, which varies with the degree of ventilation of the dwellings, depends on the buildings' construction 2,3,17 . Therefore, energy-saving house insulation tends to increase the indoor humidity and may lead to higher house dust mite densities (especially D. pteronyssinus) 4,10,26 .
Mites of the families Glycyphagidae and Acaridae are considered much more sensitive to desiccation than pyroglyphids 4,5,12,16,35 . It was also suggested that some domestic mites thrive in very damp conditions. This group includes acarids, glycyphagids and cheyletids. Therefore, the presence and abundance of these mites can be usually used as an indicator of humid environments 4,9,12,16,29 . In general, these mites are not as abundant and frequent in Europe as in the Tropics 6,7,9,10,12,16,29 .
The influence of different housing conditions (both biotic and abiotic) on dust mite populations was widely reviewed by Hart 1 . Some of them such as type of heating, type of mattress, age of furniture, type of bedding, carpets, soft furnishing, soft toys, age of house, number of occupants, presence of pets, floor heating, may influence dust mite populations [1][2][3]5,7 . Certain recent studies, however, showed weak or no correlation between these environmental factors and mite density 4,9,12,26 .
Environmental factors proved to be significant for the intensification of allergy symptoms in children whose flats were included in the study. A negative correlation between intensification of symptoms and the size of a flat as well as a positive correlation with the number of family members can be associated with an increase in the number of allergens (not only mites) under specific conditions.
An increased exposure to various allergens, including mites, and as a result the intensification of asthma symptoms could have occurred as a result of increased sealing of flats by installing PVC windows, as well as the presence of plants in the flat, sleeping on a couch and using a pillow (significant correlation with asthma severity). Our results are consistent with numerous studies, where the relationship between airborne allergens, air pollution and the development and intensity of bronchial asthma symptoms were found 18,29,36,37 . Furthermore, studies assessing the influence of prevention on the reduction of bronchial asthma symptoms confirm the findings resulting from the obtained data 18,38,39 . At the same time, there are reports showing that prevention based only on the reduction of exposure to mite allergens in the household environment does not provide asthma patients with significant clinical benefits 16,[22][23][24][39][40][41] .
Rhinitis was significantly associated with the number of D. farinae, total house dust mites and total domestic mites per gram of dust. Additionally, the intensity of allergic rhinitis was correlated with environmental conditions. A greater intensity occurred in children living in newer free standing buildings with a lower bedroom temperature, and in buildings with PVC windows, which is certainly related to their greater insulation and the higher level of humidity in rooms; both factors facilitate the reproduction of mites. A lower temperature in bedrooms of children participating in our study was a factor promoting intensification of allergic rhinitis. Two conditions should be met for intensive mite development, one being high temperature (> 20 °C) and the other high humidity (80% of relative humidity). However, it is difficult to compare it with our results as the humidity in the rooms was not measured. Greater intensity of allergic rhinitis was found in children living in free standing buildings. Considering the observations on the behavior of children living in the city, it can be concluded that these children had the opportunity to spend more time outside, in the vicinity of their houses. This kind of behavior promotes the exposure to air pollution, especially fumes released into the atmosphere from car engines. www.nature.com/scientificreports/ studies have confirmed this mechanism of rhinitis induction and intensification 42,43 . Thus, this relationship does not necessarily have to be induced by the household allergens, including mites. The intensification of atopic dermatitis was positively correlated with inhabiting free standing buildings and the type of windows installed (e.g. PVC). Window insulation (PVC) increases the exposure to household allergens, including house dust mites. According to literature, airborne allergens, including household ones, can intensify dermal lesions in atopic dermatitis patients [43][44][45][46][47][48][49][50] .
As described above, in accordance with the authors' own materials, the presence of PVC windows was also significant for the severity of asthma and the occurrence of allergic rhinitis. Numerous correlations of the intensity of allergy symptoms with the seasons (particular months) are difficult to explain only by the household environment condition. The relationships between the concentrations of IgE against D. pteronyssinus and D. farinae, positive correlations with the existence of insulated PVC windows in flats and negative correlations with a place to sleep, deserves special attention. Children sleeping on sofas showed higher IgE concentrations. Moreover, older buildings and stoves are more favourable for the occurrence of both D. pteronyssinus and the domestic non-pyroglyphids, whereas new buildings, with central heating systems, for the higher abundance of D. farinae. It should also be stressed that D. farinae was usually more abundant per gram of dust in samples from couches and sofas whereas D. pteronyssinus in bed mattresses 6,7,9 .

Conclusions
The severity of bronchial asthma in the examined children was associated with those environmental factors influencing the species composition and density of domestic mites in the said children's dwellings. The relationships also affect other allergic symptoms coexisting with asthma, such as atopic dermatitis and rhinitis. Our attention should be directed to the differences in the influence of particular environmental factors (both abiotic and biotic) on the occurrence and abundance of D. pteronyssinus and D. farinae in the dwellings of allergic patients. These factors should also be taken into account during medical interviews.
Mite prevalence and density in the examined dwellings suggests associations between density of some mite taxa (per gram of dust) and the following both abiotic or biotic indoor environmental factors: type and age of building (D. pteronyssinus with old brick houses, D. farinae with block houses and new houses), type of heating (D. pteronyssinus and cheyletids with coal-stoves), presence of pets (positive influence on densities of domestic or total mites and live D. pteronyssinus), family size and flat sizes (positive influence of smaller families or flats), suburban localisation (on D. farinae), place of sampling (beddings and beds on pyroglyphids, domestic and total mites), higher weight of sample (with exception of live D. farinae), type of sleeping accommodation (mattresses on live pyroglyphids and couches on D. farinae, total pyroglyphids, total domestic mites or total mites), absence of flowers/plants (on live D. farinae), lower level/floor of a flat (on D. farinae, L. destructor, total pyroglyphids, total domestic mites and total mites), type of windows (PVC on D. farinae and wooden on live pyroglyphids), higher cleaning frequency and higher moisture. Generally, older buildings and stoves are more favourable for the occurrence of both D. pteronyssinus and the domestic non-pyroglyphids, whereas new buildings, with central heating systems, for the higher abundance of D. farinae. It should be also stressed that D. farinae was usually more abundant per gram of dust in samples from couches and sofas whereas D. pteronyssinus in bed mattresses.
The obtained results regarding domestic acarofauna in the examined dwellings of asthmatic subjects cannot completely account for all variables determining the severity of asthma, as those persons may also be exposed to other indoor allergens. On the other hand, they are also exposed to mite allergens in different outdoor environments; however, these environments are usually home to other mite taxa, not only for the species of the family Pyroglyphidae, known as the house-dust mites. The actual study was performed in 16 flats occupied by 16 patients suffering from bronchial asthma which have agreed to participate in the study. This limited the number of flats examined. As the occurrence and density of mites in dust samples from different places or dwellings may vary to a considerable extent and the number of actually examined flats was limited, the further and more extensive studies are highly desirable. Above all these studies are needed to better clarify the relationships between indoor environmental factors, symptoms of asthma and the species composition of domestic mites from dwellings and other public places in Poland.