Humid indoor environments may be colonised by allergenic filamentous microfungi (moulds), Aspergillus spp., Penicillium spp., Cladosporium spp., and Alternaria spp. in particular. Mould-induced respiratory diseases are a worldwide problem. In the last two decades, mould allergens and glucans have been used as markers of indoor exposure to moulds. Recently, mould allergens Alt a 1 (Alternaria alternata) and Asp f 1 (Aspergillus fumigatus) have been analysed in various environments (residential and occupational) with enzyme-linked immunosorbent assays, which use monoclonal or polyclonal antibodies. Household Alt a 1 and Asp f 1 levels were usually under the limit of the method detection. By contrast, higher levels of mould allergens were found in environments with high levels of bioaerosols such as poultry farms and sawmills. Data on allergen Alt a 1 and Asp f 1 levels in agricultural settings may provide information on possible colonisation of respective moulds and point out to mould-related diseases in occupants.
Dust mites, cockroaches, and pets (cats, dogs) are common in homes worldwide, and many species are the source of potent allergens which cause allergic diseases. These diseases are influenced by genetic predisposition and environmental exposure. Generally, the levels of house dust mite (Der p 1 and Der f 1) and cockroach (Bla g 1, Bla g 2) allergens are used as markers of indoor exposure to arthropods.
This article reviews the findings of allergens Der p 1, Der f 1, and Bla g 1 in randomly selected urban households in Zagreb (Croatia) measured from 2006 to 2010 and compares them with exposure to arthropod allergens in other countries. In short, house dust mite allergen levels in Croatian homes are low, but exposure is common; Der p 1 was found in 73 % and Der f 1 in 83 % of the households. By contrast, exposure to cockroach allergen Bla g 1 was both low and uncommon (13 %). Exposure to multiple allergens associated with sensitisation and asthma was not frequent in urban homes in Croatia. However, further studies should include monitoring of both arthropod and pet allergens in high-risk populations in inland and coastal Croatia. They should also investigate a complex dose-response relationship between exposure and sensitisation/asthma development, especially in early childhood.
Bla g 1 Allergen Levels in Zagreb Area Household Dust
Cockroach allergy is a health problem in many parts of the world. In urban environments, indoor exposure to cockroach allergens involves a risk of asthma. The aim of this study was to measure the mass fraction of Bla g 1, a major allergen of the German cockroach (Blatella germanica) in 30 house samples, collected at random from Zagreb area households, Croatia. Dust samples were collected on cellulose filters by vacuuming living rooms floors. After extraction, Bla g 1 was detected using the commercial enzyme-linked immunosorbent assay (ELISA). Only four of the thirty households had detectable Bla g 1 levels, and only in one was its concentration higher than 2.0 U g-1, the threshold associated with sensitisation. The Bla g 1 ELISA proved highly sensitive, with the detection limit of 0.12 U g-1. The within- and between-assay imprecision was 8.9 % and 14.4 %, respectively, and accuracy 85 % to 120 %. Low Bla g 1 levels in the household dust support previously reported low prevalence of skin sensitisation to B. germanica among Zagreb residents. Further monitoring should reveal if there are differences in cockroach allergen exposure and sensitisation between households from other geographic areas in Croatia.
Ljerka Prester, Veda Marija Varnai and Jelena Macan
The CD14 receptor is expressed on the surfaces of monocytes, macrophages, and, to a lesser extent, of activated granulocytes and B lymphocytes (mCD14). It also exists in soluble form in the serum (sCD14), in which it binds microbial compounds and indoor allergens. The objectives of this study were to see whether serum sCD14 concentrations could be used as a marker of atopic disorders and to estimate the effects of environmental factors (tobacco smoke exposure, childhood residence in urban or rural areas, and having a pet) on sCD14 and IgE values. Mass fraction of sCD14 and total IgE were determined in the sera of atopic (N=53) and non-atopic (N=35) participants using the standard enzyme-linked immunosorbent assay. Exposure to pets and environmental tobacco smoke was estimated based on the International Study of Asthma and Allergies in Childhood questionnaire (ISAAC). Median sCD14 concentration was significantly lower in atopic than in non-atopic girls (3.49 vs. 3.83 μg mL-1; p<0.010). The number of smokers at home positively correlated with the sCD14 level in atopics, and urban atopics had significantly lower sCD14 levels than rural atopics (3.47 vs. 3.92 μg mL-1, p=0.028). Median total IgE concentration was significantly lower in atopic pet owners than in atopics with no pets (161 vs. 252 kIU L-1; p=0.021). In conclusion, while sex and environmental factors might be involved in sCD14 expression, particularly in atopics, we found no correlation between sCD14 and total IgE concentrations. The usefulness of sCD14 as a marker of atopic disorders should be investigated further, particularly in relation to the severity of allergic disorders.
Determination of mite Allergens in House Dust Using the Enzyme Immunoassay
The aim of this study was to determine the level of two major mite allergens Dermatophagoides pteronyssinus (Der p 1) and Dermatophagoides farinae (Der f 1) in 30 urban homes in Zagreb, Croatia, using the enzyme immunoassay with two monoclonal antibodies which has been established as the reference method for indoor allergen analysis. Dust samples were taken by vacuuming a carpeted area and collected on cellulose filters. The ranges of Der p 1 and Der f 1 were (0.1-12.5) μg g-1 (median 0.32 μg g-1) and (0.1-31.2) μg g-1 (median 0.35 μg g-1), respectively. Der p 1 and Der f 1 (>2 μg g-1) associated with increased risk of sensitization to mite allergens were found in approximately 16% homes for each allergen. The sum of allergen (Der p 1 + Der f 1) exceeded the lower threshold in 27% of homes. Analytical evaluation of the ELISA assay showed satisfactory results for precision (intra-assay CV <6.9%, inter-assay CV<13.3%), accuracy (91% to 93%), and sensitivity (2 ng mL-1).
The ELISA assay for the measurement of dust mite allergens demonstrated very good analytical characteristics for routine laboratory use, and will provide the essential basis for our future studies of various indoor allergens.
Der p 1 is the main allergen of house dust mite Dermatophagoides pteronyssinus, which has routinely been detected in residential dust. However, the procedure for extracting Der p 1 from reservoir dust has not been well defi ned. The aim of this study was to compare Der p 1 mass fractions in dust extracts prepared using the following extraction buffers: phosphate (pH 7.4), borate (pH 8.0), and ammonium bicarbonate (pH 8.0), all with 0.05 % Tween 20. Twenty-eight dust samples were divided into three aliquots and each portion was extracted with one of the three buffers at room temperature. Der p 1 mass fractions were measured in a total of 84 dust extracts using the enzyme immunoassay (range: 0.1 μg g-1 to 7.53 μg g-1). Statistical methods including intraclass correlation showed a high agreement between Der p 1 mass fractions irrespective of the extracting medium. Our results suggest that all three buffers are suitable for the extraction of mite allergens and routine Der p 1 analysis in dust.
Ljerka Prester, Jelena Macan, Kristina Matković and Marija Vučemilo
Determination of Aspergillus Fumigatus Allergen 1 in Poultry Farms Using the Enzyme Immunoassay
Poultry farms contain high levels of allergenic fungi, and Aspergillus spp. is the most common genus of moulds. Aspergillus fumigatus antigens are responsible for the development of several respiratory diseases including asthma. The aim of this study was to measure the mass fraction of Asp f 1, a major allergen of Asperillus fumigatus in 37 indoor dust samples collected from four poultry farms in a rural area of the Zagreb County (Croatia) using the enzyme-linked immunosorbent assay. More than 62 % of dust samples had detectable Asp f 1 levels (limit of detection 3.6 ng g-1). The overall mean Asp f 1 level was 17.9 ng g-1 [range (3.8 to 72.4) ng g-1]. Satisfactory results were obtained for analytical within-run imprecision (6.7 %), between-run imprecision (10.5 %), and accuracy (91 % to 115 %). Microclimate parameters (air temperature, relative humidity, and velocity) were within the recommended ranges in all poultry farms. This study has shown that Asp f 1 settles on dust at poultry farms and that occupational exposure to this allergen deserves monitoring in livestock buildings.
Ljerka Prester, Tatjana Orct, Jelena Macan, Jelena Vukušić and Dubravka Kipčić
Determination of Biogenic Amines and Endotoxin in Squid, Musky Octopus, Norway Lobster, and Mussel Stored at Room Temperature
Little research has been published on the indicators of spoilage in Mediterranean molluscan shellfish and crustaceans. Thus is why we studied changes in the concentrations of endotoxin and four biogenic amines (histamine, putrescine, tyramine and cadaverine) in European common squid (Loligo subulata, Lamarck, 1798), musky octopus (Eledone moschata, Lamarck, 1798), Norway lobster (Nephrops norvegicus, Linnaeus, 1758), and mussel (Mytilus galloprovincialis, Lamarck, 1819) from the Adriatic Sea stored at room temperature for 24 h. Endotoxin load in fresh squid, Norway lobster, and mussel (<1 EU mg-1) indicated good microbiological quality of raw samples. Biogenic amine index (as the sum of histamine, putrescine, tyramine, and cadaverine) correlated well with endotoxin load in squid (r=0.978, p<0.001) and musky octopus (r=0.874, p<0.01). A good correlation was also found between endotoxin and putrescine in Norway lobster (r=0.777, p<0.05). The highest endotoxin load was found in decomposed mussels and was associated with histamine alone. In conclusion, increase in biogenic amine levels is species-specific. Endotoxin analysis could be used for rapid assessment of microbiological quality of cephalopods and shellfish.
Veda Varnai, Anita Ljubičić, Ljerka Prester and Jelena Macan
Exhaled Breath Condensate pH in Adult Croatian Population Without Respiratory Disorders: How Healthy a Population Should Be to Provide Normative Data?
The aim of this study was to obtain preliminary exhaled breath condensate (EBC) pH values for healthy adult Croatian subjects, and to evaluate criteria for defining respiratory health of population providing normal EBC pH values in epidemiologic studies. In 109 adults without a history of lower airway symptoms (AS), four groups were described by narrowing the definition of "health" down to 1) without lower AS; 2) without lower and upper AS; 3) without AS, with normal FEV1 and bronchial normoreactivity; 4) without AS, with normal FEV1, bronchial normoreactivity, normal total IgE, and with negative skin prick test. Median EBC pH values did not differ between the groups (7.72, 7.73, 7.73, 7.73), but as health criteria got stricter, we observed a slight, nonsignificant increase in minimal pH values (6.95, 7.10, 7.20, 7.37). Median EBC pH values with interquartile range in the total sample (7.72; 7.63 to 7.76) were within the range previously reported by other authors. They did not differ regarding sex, smoking habit and atopic status, and were not associated with age, FEV1 or total IgE. The non-significant trend in EBC pH observed with stricter criteria of respiratory health and atopic status indicates the need for further research on criteria for defining healthy population in a larger sample.
Ljerka Prester, Nives Protrka, Jelena Macan and Marina Katunarić
CD14 is a co-receptor involved in the recognition of Gram-negative and Gram-positive bacteria, the latter known to cause dental caries. The aim of this study was to determine whether soluble CD14 (sCD14) in saliva was associated with caries activity and the collection method from the saliva. The study included 55 participants aged 20 to 40 years, 30 with dental caries and 25 caries-free controls. We collected 110 saliva samples in total, 55 of resting saliva and 55 of mechanically stimulated saliva. Median levels of sCD14, measured with a matrix-matched enzyme-linked immunosorbent assay (ELISA), were higher in the caries-active than in the caries-free group in either resting (203.3 vs.167.9 ng mL-1; P<0.01) or stimulated saliva (201 vs.105.7 ng mL-1; P<0.01). The resting salivary flow rate was lower in the caries-active than caries-free group (0.61±0.42 vs. 0.98±0.52 mL min-1; P<0.01). Hyposalivation was observed only in the caries-active group (10 and 13 % in stimulated and resting saliva, respectively). Higher salivary sCD14 levels and secretion rates were clearly associated with dental caries and resting saliva. Future studies should focus on the clinical utility of salivary sCD14 as a potential biomarker and predictor of future caries events.