|Year : 2019 | Volume
| Issue : 1 | Page : 88-96
Morphological identification of house dust mite species in Menoufia Governorate and their antigen effect in immunoglobulin E response in allergic patients
Wafaa M El Kersh1, Mona M. K. El Sobky1, Nancy M Harbah1, Hany M Heikl2, Doaa I. M. Abou Galalah1
1 Department of Medical Parasitology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Insect, Faculty of Agriculture, Menoufia University, Menoufia, Egypt
|Date of Submission||06-Mar-2016|
|Date of Acceptance||17-Apr-2016|
|Date of Web Publication||17-Apr-2019|
Doaa I. M. Abou Galalah
Shebin El-Kom, Menoufia
Source of Support: None, Conflict of Interest: None
The aim was to identify and differentiate different species of house dust mites (HDMs) from dust samples from different areas in Menoufia Governorate, with preparation of antigen from each HDM identified species for estimation of specific immunoglobulin E (IgE) and total monoclonal IgE in selected allergic patients.
The HDMs are considered the commonest sources of airborne allergens worldwide. Diagnosis of HDM allergy is a challenge, which is reflected in the treatment and prophylaxis of allergy.
Materials and methods
Dust samples were collected from houses in different areas in Menoufia Governorate. Mites were isolated and morphologically classified, and crude antigens were prepared from each identified species. A total of 42 allergic patients were selected, in addition to 10 control healthy individuals. Total and specific HDM monoclonal IgE levels were measured by conventional enzyme-linked immunosorbent assay technique.
Rural areas showed higher density of HDM, with a higher significant difference than urban areas, Melig was the highest infested rural area, with significant difference. The predominant species were Dermatophagoides pteronyssinus (72.6%), Dermatophagoides farina (51.2%), and Tyrophagus putrescentiae (42.6%). Total monoclonal IgE estimation revealed high levels in all studied patients. D. pteronyssinus antigen showed highly significant difference in diagnosis of 95% of patients with asthma. Moreover, D. farina antigen showed significant difference in diagnosis of 90.9% of patients with atopic dermatitis, whereas T. Putrescentiae antigen showed high rate (72.7%) in patients with atopic dermatitis although insignificant difference in the diagnosis of different allergic patients.
D. pteronyssinus represented the most common HDM species in dust samples detected in selected areas, whereas D. pteronyssinus and D. farina antigens are the most common sensitizing HDM antigens in the studied patients. The use of these antigens in diagnosis, vaccination, and hyposensitization is recommended.
Keywords: allergy, Dermatophagoides pteronyssinus and Dermatophagoides farina, enzyme-linked immunosorbent assay, house dust mites, immunoglobulin E
|How to cite this article:|
El Kersh WM, El Sobky MM, Harbah NM, Heikl HM, Abou Galalah DI. Morphological identification of house dust mite species in Menoufia Governorate and their antigen effect in immunoglobulin E response in allergic patients. Menoufia Med J 2019;32:88-96
|How to cite this URL:|
El Kersh WM, El Sobky MM, Harbah NM, Heikl HM, Abou Galalah DI. Morphological identification of house dust mite species in Menoufia Governorate and their antigen effect in immunoglobulin E response in allergic patients. Menoufia Med J [serial online] 2019 [cited 2019 May 21];32:88-96. Available from: http://www.mmj.eg.net/text.asp?2019/32/1/88/256145
| Introduction|| |
House dust contains the most widespread source of indoor allergens, namely, house dust mites (HDMs) . The HDMs are considered the commonest sources of airborne allergens worldwide .
Wharton  considered HDMs to be a member of subclass Acari of subphylum Chelicerata. They belong to class Arachnida, order Acariformis. These diminutive creatures affect a sizeable worldwide population and are the predominant source causing allergic pathological states .
There are approximately 16 genera and 46 species of HDMs. The most prevalent in the dust environment are members of the family Pyroglyphidae. The most common members associated with allergic diseases are Dermatophagoides pteronyssinus, Dermatophagoides farinae, D. microceras, and Euroglyphys maynei ,.
The HDMs are invertebrates, as they do not have an internal skeleton. They are free living but have many anatomical features in common with parasitic mites. They are very tiny arachnids that inhabit houses in beds and living rooms, feed on sloughed skin scales, and thrive in warm, dark, and humid atmosphere . They cause disease by themselves and also by excretion of fecal pellets rich in proteolytic enzymes that are very allergenic to human, which are the main cause of indoor allergies .
Atopic patients exposed to HDM allergens develop potent inflammatory diseases and produce different grades of immunologic responses that lead to different types of allergy such as bronchial asthma, atopic dermatitis (AD), and allergic rhinitis ,,.
Diagnosis of HDM allergy is usually carried out by history, eosinophilic count, skin prick test, and total and specific HDM serum immunoglobulin(Ig)E detection .
Treatment of HDM allergy includes corticosteroids and anti-inflammatory drugs. Hyposensitization using vaccines also plays a role in treatment of allergy .
Specific immunotherapy treatment involves the repetitive application of an allergen by subcutaneous injection or sublingual application. This approach modifies the natural course of the asthmatic response and alleviates the severity of patient and asthma symptoms .
The aim of this study was to identify the most predominant HDM species in different areas in Menoufia Governorate, preparation of HDMs antigen from each identified species, and examination of total monoclonal IgE in addition to specific IgE enhancement by prepared antigens in different allergic patients.
| Materials and methods|| |
This study was approved by the ethical committee of faculty of medicine, Menoufia University. This study was carried out in different areas in Menoufia Governorate, including urban areas (Shebin El-Kom, Quesna, and Ashmon) and rural areas (Melig, Battanon, and Shanawan).
Collection of house dust samples
A total of 282 house dust samples were collected monthly throughout the period from June 2012 to August 2013. Dust samples were obtained from the bedrooms, mattresses, floor, bedding, living rooms, and kitchens. One square meter of each place was vacuumed for 1 min. Samples were put in plastic bags and carefully tied. Each sample was given a numerical number and then stored at 4°C to avoid proliferation, and examined in the laboratory within 24 h of collection .
Examination of dust samples
Mites were extracted from dust samples using a modified-Berlese funnels in Insect Department, Agriculture Faculty, Menoufia University . Samples were left on the funnels for 48 h to be separated in a glass jar. After the extraction period, the contents of the glass jar were transferred to Petridish for examining under the stereomicroscope, and each identified species was isolated in separate beakers. Each mite sample was prepared by temporary and permanent methods .
Identification of house dust mites
The temporary preparation method
Using a concave slide, a small drop of lactic acid was put in the medium of concave area for clearance of mite specimens. The slides were directly examined under the stereomicroscope .
The permanent preparation method
A small drop of Hoyer's medium (50 μm) was put on the mite specimens in the center of a clean glass slide. This process was used for clearing and fixation of the mite. The slides were microscopically examined after drying of Hoyer's medium .
Mite species were examined and identified according to the keys given by Van Bronswijk and Sinha , Arlian , and Colloff . Marks of species identification are summarized.
The HDMs were separated according to species, the data were collected, and in small beakers containing saline, they were kept frozen for preparation of antigen .
This procedure was performed in Theodor Bilharz Research Institute. Aqueous solution of crude body extract antigen of HDM species was prepared according to Rubaire-Akiiki and Mutinga . In brief, the method was performed by washing of collected mites three times in distilled water. After homogenization, the extracts were centrifuged, and the supernatants were collected and sterile-filtered (0.22 mm) into sterile vials for storage at 4°C. The total protein concentration of each extract was determined using the Bradford protein assay with bovine serum albumin as the standard . Before using the antigen, it was resuspended in sterile PBS solution in concentration of 0.2 mg/ml.
This study was carried out on 52 persons aged from 12 to 50 years old. They were divided into 42 allergic patients (group 1) (18 males and 24 females) and 10 apparently healthy nonallergic individuals (group 2) (three males and seven females) with no history of atopy as control individuals. The selected patients were attending different clinics, ENT, Chest, and Dermatology outpatient clinics, of Menoufia University Hospital and Shebin El-Kom Educational Hospital. According to positive symptoms, they were selected and divided into the following groups: asthmatic group (20 patients with asthma with positive chest symptoms), AD group (11 patients with AD with positive atopic skin symptoms), and allergic rhinitis group (11 patients with allergic rhinitis with positive upper respiratory symptom). The selected patients and control were clinically examined and were given questionnaires to be filled in the clinical data sheet. Peripheral blood samples (3 ml) from all studied subjects were collected after taking written consents, and serum samples were separated, labeled, and kept at –20°C.
Detection of monoclonal serum IgE 
Enzyme-linked immunosorbent assay (ELISA) was used to determine the serum concentration of total monoclonal IgE in selected patients and controls using commercially available monoclonal anti-IgE kits (Human GmbH.65205, Wiesbaden, Germany). The optical density values were determined by reading the plate at wavelength of 450 nm using ELISA reader spectrophotometer; it was measured within 30 min after termination of the reaction. Normal level for total IgE was less than 25 IU/ml.
Detection of HDM-specific IgE 
ELISA technique using previously prepared HDM antigens was performed for all allergic patients and controls, where 100 μl of carbonate–bicarbonate buffer containing the prepared crude antigen of each type of HDM was added in each well of microtiter plate (1 ml of antigen and 0.5 ml carbonate–bicarbonate buffer). The plate was incubated at 4°C overnight and then the buffer was removed into decontaminating pan, and the plate was washed three times with PBS Tween 20 (5 min for each). Serum samples were diluted 1:500 in a master plate by diluting buffer, and 100 μl of the diluted serum was transplanted in their corresponding well. The plate was incubated for 60 min at 37°C. The plate had been washed three times. The conjugate solution (Human GmbH.65205) (100 μl) was added to each well, and the plate was incubated for 60 min at 37°C. After washing the plate three times, 100 μl of the substrate working solution was added to each well, and the plate was placed in dark place for 30 min. A volume of 5 μl of the stopping solution was added to each well to stop the reaction. The positive result for ELISA was considered when the optical density of the tested sample is double the mean of the negative control.
Data were collected, tabulated, and statistically analyzed by computer using statistical package for the social science software (SPSS) version 11 by using (Released 2011, IBM SPSS Statistics for Windows, Version 2010; SPSS Inc., Chicago, Illinois, USA). P values of less than 0.05 were considered statistically significant.
| Results|| |
Collected HDMs from different areas in Menoufia Governorate
Of 282 collected dust samples from houses in six areas in Menoufia Governorate, 202 (71.6%) samples were positive for HDMs. The highest positive HDMs samples were in Melig (90.2%) and the lowest positive samples were in Quiesna (40%), with significant difference (P < 0.05) [Table 1].
|Table 1: Distribution of house dust mites in rural and urban areas in Menoufia Governorate|
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There was statistically significant higher positivity of HDMs in dust samples from rural area (61.7%) than those from urban areas (38.3%) [Table 1]. The results also showed that most house dust samples contained different types of mites, and few samples showed only one type. The predominant species in all examined areas were D. pteronyssinus (72.6%) followed by D. farinae (51.2%) and storage mite, Tyrophagus putrescentiae (42.6%) [Table 2].
|Table 2: Distribution of house dust mites species in collected house dust samples|
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Morphological identification and classification of the detected HDMs
After isolation, preservation, and mounting of different HDMs, species identification was carried out according to Van Bronswijk and Sinha , Arlian , and Colloff  [Table 3]. Isolated D. farina female showed less arched epigyna and short spine of last tarsus of the first leg [Figure 1] and [Figure 2]a, [Figure 2]b, but D. pteronyssinus isolated female appeared with its more arched epigyna and long spine of last tarsus of the first leg [Figure 2]c and [Figure 2]d. Regarding adult D. farina isolated male, V or Y shaped epimers of coxa 1 with large first pair of legs relative to the rest of the legs [Figure 3]a were the most obvious features, whereas in D. pteronyssinus isolated male, their epimers of coxa 1 did not form V or Y shape, with first pair of legs were not enlarged [Figure 3]b. Moreover, anal suckers were characteristic in both species. For T. putrescentiae, there were dense setae in dorsal surface with presence of vulva, epigyna, and bursa copulatrix in females [Figure 4]a and anal suckers in males [Figure 4]b.
|Table 3: Summerization of the most important identification marks between genus D. pteronyssinus, D. farinaeand T. putrescentia (Bronswijk&Sinha, 1971  and Arlian, 1989) |
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|Figure 1: (a and b) Dust samples showing house dust mite before isolation (×400).|
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|Figure 2; Adult isolated house dust mite female. (a) Adult Dermatophagoides farina female isolated from dust sample showing less arched epigyna (green arrow) and short spine of last tarsus of the first leg (red arrow) (×100). (b) High magnification of Dermatophagoides farina last tarsus of the first leg showing short spine (×400). (c) Adult Dermatophagoides pteronyssinus isolated female showing more arched epigyna (black arrow) and long spine of last tarsus of the first leg (red arrow) (×100). (d) High magnification of adult Dermatophagoides pteronyssinus last tarsus of the first leg showing long spine (×400).|
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|Figure 3: Adult male of house dust mite of different species. (a) Adult Dermatophagoides farina isolated male showing V- or Y-shaped epimers of coxa 1 (red arrow) and anal sucker (green arrow) with large first pair of legs relative to the rest of the legs (black arrow) (×100). (b) Adult Dermatophagoides pteronyssinus isolated male showing epimers of coxa 1 do not form V or Y shape (red arrow) and anal sucker (blue arrow), with first pair of legs not enlarged (green arrow) (×100).|
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|Figure 4: Adults of Tyrophagus putrescentiae. (a) Adult isolated female Tyrophagus putrescentiae showing dense setae in dorsal surface (red arrows) with presence of vulva, epigyna, and bursa copulatrix (blue arrow) (×100). (b) Adult isolated Tyrophagus putrescentiae male showing dense setae in dorsal (red arrows) surface with presence of anal suckers (blue arrow).|
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Moreover, the isolated HDM nymph showed four pairs of legs without genital organs [Figure 5].
|Figure 5: Isolated house dust mite nymph showing no genital organs (×100).|
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Results of protein content of prepared antigens
The HDMs isolated species from the dust were used for antigen preparation, and three antigens were prepared: Ag 1 (D. farina antigen), Ag 2 (D. pteronyssinus antigen), and Ag 3 (T. putrescentiae antigen).
The protein content was higher in Ag 1 (0.849 gm/ml) than of Ag 2 and Ag 3 (0.226 and 0.575 g/ml, respectively).
Analysis of most important clinical data in selected allergic patients
Regarding age, 26.2% were 12–18 years old and 73.8% of patients were greater than 18 years old. Regarding sex, most patients with asthma were female (70%), whereas most patients with AD were males (63.6%), with nearly equal percentage in patients with allergic rhinitis (54.5% in females and 45.4% in males). Previous history to allergy was recorded in 90.5% of all allergic patients with 100% in patients with allergic rhinitis [Table 4].
Family history of allergy was found in 47.6% of studied allergic patients, mainly in patients with AD (63.6%). There was seasonal variation in attacks of allergy; the attacks of 95% of patients with asthma were in winter, whereas of 81.8% of atopic patients were in autumn with significant difference. Regarding residence, 72.7% of allergic rhinitis and 70% in patients with asthma were living in rural areas, whereas 45.5% of patients with AD were from urban residence.
Results of total serum monoclonal IgE
All selected allergic patients (42) recorded higher serum level of total monoclonal IgE than normal value (25 IU/ml). It was noticed that mean IgE level for patients aged 12–18 years old was higher than that for patients aged more than 18 years old (391 and 81.3 IU/ml, respectively) [Table 5].
|Table 5: Age distribution among positive monoclonal immunoglobulin E in selected allergic patients|
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Results of specific IgE ELISA test by prepared HDM antigens
Use of Ag 2 (D. pteronyssinus) revealed higher positivity with high significant difference (P < 0.01) in diagnosis of 95% of patients with asthma, whereas lower positivity appeared by using Ag 3 and Ag1 (50 and 45%, respectively). In diagnosis of patients with AD, in spite of high significant positivity of Ag1 (90.9%), use of Ag3 and Ag2 gave lower positive rate (72.7 and 54.5%, respectively). The relation was insignificant (P > 0.05) in comparison of the three used antigens in patients with AD. Regarding Ag3, patients with AD recorded the highest positivity detection (72.7%) compared with patients with asthma and allergic rhinitis (50 and 36.4%, respectively). This positivity was insignificant (P > 0.05) [Table 6].
|Table 6: Results of enzyme-linked immunosorbent assay-immunoglobulin E for the three antigens in studied allergic patients|
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| Discussion|| |
Dominance of HDMs in dust samples was reported from different parts of the world . The present study was carried out in Menoufia Governorate and revealed that the higher density of HDM was in rural areas (81.7%), mainly in Melig village (90.2%), than urban one (52.1%), with high significant difference. The lowest infestation was in urban area, Quiesna city (40%).
These results are consistent with the results of another study in Menoufia Governorate which reported that a rural area, Melig, had the highest density of HDMs . Similar observations were reported in Menoufia Governorate by Faheem et al.  who found high density and prevalence of mites in a rural area, Bakhate, in which the economic standard was very low with high relative humidity. However, they found mites in dust samples of the offices of medical staff in relatively high intensity, and they correlate this observation to the presence of carpets, which are a good environment for living of mites. On the contrary, El-Shazly et al.  found that the prevalence of HDMs in urban areas was 59.9% and in rural areas was 46.3% in Dakahlia Governorate.
In the present study, the predominant species in all examined areas was D. pteronyssinus (72.6%) followed by D. farinae (51.2%) and lastly T. putrescentiae, which was detected in dust with a percentage of 42.6% in dust samples. These results are in accordance with Heikal  who found that of the total collected mites (5276) from different houses in Elkom Elakhdar village and Shebin El-Kom in Menoufia Governorate, the highest dominant species was D. farinae (66.1%), followed by D. pteronyssinus (23.3%), whereas the percentages of the rest species such as Chortoglyphus arcuatus, Lepidoglyphus destructor, Glycyphagus domesticus, Gohieriafusca, T. putrescentiae, Caloglyphussp, Cheyletus malaccensis, Blomia spp. and A. siro ranged between 0.16 and 2.0%. The predominance of Dermatophagoides spp. was also reported by other authors, such as Gamal-Eddin et al.  who recorded them in Tanta City in addition to other HDMs, such as T. putrescentiae, Cheyletus malaccensis, Blomia kulagini, and Acheles gracilis.
Similar results were also obtained by Kenawy et al.  and Hossny et al.  who found that D. pteronyssinus and D. farina were the most prevalent strains.
In the current work, most allergic patients were older than 18 years (73.8%), and 26.2% were between 12 and 18 years of age. Most of the patients with asthma were females (70%); however, most patients with AD were males (63.6%). Similar observations were reported by Musafiri et al.  who found that 51.9% of patients with asthma were females and 48.1% were males. Moreover, the prevalence rates of atopy were 36.4% in men aged less than or equal to 60 years versus 26.2% in men aged greater than 60 years and 30.6 versus 18.1% in women, respectively. In patients with allergic rhinitis older than 60 years, the prevalence rates were 13.0% for men and 15.4% for women, and in asthma, they were 6.6 versus 7.6%, respectively. Moreover, Wüthrich et al.  recorded that the prevalence of both rhinitis and asthma was higher in persons younger than 60 years.
Previous and family histories of allergy were also recorded as they were 90.5 and 47.6%, respectively, which is in accordance with Doshi and Tripathi , who reported that family history of allergic disorders was present in 60.8% of allergic children and 65.3% had a family history of atopy.
There was a significant difference (P < 0.05) regarding seasonal variation in attacks of allergy, where 61.9% of allergic patients showed their attacks in winter, whereas 38.1% showed their attacks in autumn. This may be owing to increased exposure to allergens and common cold attacks during winter. This is in line of Mimura et al. , who found that specific IgE scores for cedar pollen and D. pteronyssinus were significantly higher in the winter and autumn. These results may be contributed to that HDM allergens have an effect on the level of airway hyperresponsiveness in patients with allergic condition .
Regarding the results of measuring serum level of total monoclonal IgE, all selected allergic patients recorded higher IgE levels above normal range (<25 IU/ml). The mean concentration of total IgE in patients aged 12–18 years was higher (391 IU/ml) than that in patients aged more than 18 years (81.3 IU/ml).
These results were in line with Podder et al.  who found that the mean concentration of total serum IgE in allergic patients was 369 ± 26.51 IU/ml. Moreover, Ching et al.  recorded significant high concentration of serum total IgE in allergic groups as compared with the control. Milian and Diaz  reported that high level of total IgE could be contributed to modulation of the immune response by HDM allergens, causing activation of Th2 cells and B cells, which stimulate an IgE response. Similarly, a significant increase in total IgE and IgG in the serum of HDM-challenged mice compared with allergen-naïve mice was recorded .
In the present work, specific IgE by the prepared three antigens (D. farina, D. pteronyssinus, and Tyrophagus, respectively) in sera of studied allergic patients showed that using Ag 2 showed highly significant difference (P < 0.01) in diagnosis of 95% of patients with asthma, whereas lower positivity was recorded in Ag 3 (50%) and Ag1 (45%). These results are in agreement with Duro et al. , who found that 100% of respiratory allergic patients were positive to D. pteronyssinus, whereas 70% were positive to D. farina. Moreover, Podder et al.  revealed that the responses of patients with asthma to HDM allergen, D. pteronyssinus, B. tropicalis, and D. farina were 96.22, 71.91, 90, and 88.76%, respectively, by specific IgE antibody skin prick test.
The use of three antigens in diagnosis of patients with AD and allergic rhinitis showed high positive results of 90% by Ag1 and 72.7% by Ag3, respectively, with insignificant differences. In contrary to our results, Kim et al.  recorded that 100% of patients (allergic asthma, AD, and allergic rhinitis) were sensitized to D. farina antigen, parallely 98.8% had detectable serum IgE to D. pteronyssinus, with significant differences between different types of allergy.
The present results show that there were significant differences (P < 0.05) in using Ag1 and Ag2 in diagnosis of different allergic patients, especially AD and asthmatic cases, correspondingly.
These results were in line with Zhao et al.  who revealed that exposure to allergens from D. farinae and D. pteronyssinus (Ag1 and Ag2, respectively) is associated with various allergic diseases such as asthma. More than 80% of allergic patients sensitized to HDM have IgE antibodies against D. farinae and D. pteronyssinus allergens. Lieo et al.  found that the level of T. putrescentiae-specific IgE in sensitized mice increased significantly compared with the normal group.
On the contrary, Lâm et al.  and Ramírez-Heredia et al.  reported that the most common sensitizer among adults in northern Vietnam was the storage mite (B. tropicalis) followed by D. pteronyssinus and D. farinae. These variation between these results could be contributed to the difference in environment, temperature, humidity, and types of mites isolated, especially the species of storage mites.
These results are in parallel to the detection of D. pteronyssinus and D. farina as the most common sensitizing HDM in this area of study ,. It was concluded that these antigens could be used in diagnosis, vaccination, hyposensitization and immunotherapy, and we need further studies on their use.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]