Exposure and sensitization to dust mite allergens among asthmatic children in Sao Paulo, Brazil

ctinical and Experimental Allergy, 1991. Volume 21. pages 433-439 ADONIS 095478949100062Z Exposure and sensitization to dust mite allergens among asthmatic children in Sao Paulo, Brazil L K ARRUDA, M. C. RlZZOf, M. D. CHAPMAN, E, FERNANDEZCALDAS*, D. BAGGIOJ, T, A, E. PLATTS-MILLS and C. K. NASPITZt University of Virginia. Charlottesiilte. VA. *Univer.sity of South Ftorida, Tampa. FL. U.S.A., -fPaulista Sclwot of Medicine, and XUniversity of Sao Paulo. Sao Paulo. Brazil Summary A group of 20 mite allergie asthmatic children aged 6-12 years old. living in Sao Paulo, Brazil, was studied regarding their degree of sensitization to house dust mites and exposure to mite allergens in their homes. In 18 out of 20 houses at !east one dust samp!e was obtained which contained > !0 /ig Der p I/g of dust. The highest !evels of Dermatophagoides pteronyssinus a!lergens, Der p I and Group II, were measured in bedding samples (geometric mean 38-4 and 36 6 /ig/g, respective!y), fo!!owed by bedroom floor, TV room and ltitchen. Mite al!ergen !evels in Brazilian houses were as high as those reported to be associated with sensitization and acute attaeks of asthma in other parts of the world. In !<eeping with previous reports that D. farinae is rare!y found in Brazi!, Derf\ was undetectable or found in very !ow leve!s ( < 0 5 /ig/g). Leve!s of cat allergen Fel dl of >S /ig/g of dust were obtained on!y in 2 houses on!y. Cockroach a!lergen Bla g I was detected in five out of 20 houses. Leve!s of IgE antibodies to D. pteronyssinus were > 200 RAST U/ml in 19 out of 20 children (geometric mean 1588 RAST U/ml). IgE antibodies to cat, cockroach, A. fumigatus, ragweed and rye grass poUens were undetectab!e or < 80 RAST U/ml. IgE antibodies to the mite Blomia tropicalis were also measured, and leve!s > 200 RAST U/m! were observed in !3 out of 20 sera. Immunoabsorption studies demonstrated that the bu!k of the IgE antibody to B. tropicalis (64%) was to species-specific a!!ergens and that 36% were cross-reactive with D. pteronvssinus. The implication of our resu!ts is that management of chi!dren with asthma in Sao Pau!o shou!d include skin testing for aUergy to both Dermatophagoides and B. tropicalis as well as recommendations about environmenta! control of house dust mite exposure. Clinical and Experimental Allergy. Vo!. 2!, pp. 433-439. Received 3 September !990; revised 2 January !991; accepted 4 January !99!. Introduction The importance of house dust mites as a source of al!ergens and their contribution to bronchial asthma, rhinitis and atopic dermatitis has been recognized for many years [1,2]. Epidemio!ogic studies have demonstrated that the presence of IgE antibodies to dust mites and other indoor a!!ergens are major risk factors for asthma [3-5], Some of the studies have come from tropica! countries such as Zambia, Papua New Guinea and Brazi! [6,7]. The deve!opment of sensitive monoCorrespondence: L. Karla Arruda. MD. Division of Attergy and Clinical Immunology, Universiiy of Virginia Health Sciences Center, Box 225. Charlottesville, VA 2290«, U.S.A. c!ona! antibody-based immunoassays for measuring Dermatophagoides sp. Group I and group I! a!!ergens have both improved the standardization of extracts and greatly simplified the assessment of exposure to dust mite aUergens [3]. Although pyrog!yphid mites have been shown to dominate the mite fauna in house dust, IgE-mediated reactions to storage mites have been documented by skin tests, RAST and bronchial chaUenge tests [8-! 3]. These nonpyroglyphid mites include Glycyphagus domesticus and Lepidoglyphus destructor (fami!y Glycyphagidae), Tyrophagus putre.scentiae and Acarus siro (family AcaHdae). Blomia tropicalis is regarded as a storage mite, however high densities of this species have been reported 433 434 L. Karla Arruda et al. in house dust samples from several countries, including the U.S.A [14-17]. Studies carried out in Sao Paulo. Brazil, have identified D. pteronyssinus and B. tropicalis as the most frequent mite species in house dust [7]. At Paulista School of Medicine, two-thirds of the patients who are referred to the Paediatric Allergy Clinic give positive immediate skin tests to house dust mite extracts {Dermatophagoides species). A recent report from Sao Paulo on 123 adult patients with asthma and/or rhinitis showed that 77% were skin test positive to D. pteronyssinus and 78'^, were positive to B. tropicalis [18]. In populations exposed to bothmites, it is not clear how much of the skin response is specific for B. tropicalis or is shared with antigens from D. pteronyssinus. The present study was designed to quantify exposure to house dust mite allergens and IgE antibodies in a group of rnite-allergic asthmatic children living in Sao Paulo. We also investigated the antigenic relationships between B. tropicalis and D. pteronyssinus. Subjects and methods Subjects Twenty children (11 girls, nine boys) with moderate to severe asthma, aged 6-12 years old. were enrolled in the present study, from patients who attended the Paediatric Allergy Clinic of Paulista School of Medicine. Sao Paulo. Brazil. Children were selected based on positive immediate skin test to D. pteronyssinus (Dpt) and/or D. farinae (Df) extracts (Alergomed, Merck. Munich. Germany). A positive test was defined by a weal size of > 5 x 5 mm in diameter on prick test. Diagnosis and classification of asthma was done by history, physical exam and spirometry [19]. Serum samples were obtained in August to October. 1988. and patients had their houses visited for collection of house dust samples over the same period of time. B. tropicalis and D. pteronyssinus extracts Two B. tropicalis extracts were used in the present study. A whole culture extract was produced in Sao Paulo, Brazil, as follows: B. tropicalis was grown in a medium containing horse skin scales, fish fiour, beer yeast, manioc toasted flour and soybean flour. Mites, faecal particles and culture medium were defatted with ether, extracted for I hr at 4 C in saline, sonicated and centrifuged at 48 000^ for 30 min. The supernatant was dialysed against saline for 24 hr. A mite body extract was obtained in Tampa, FL, according to the following method: B. tropicalis was cultured on a medium of autoclaved nude mice food and Brine shrimp 1:1 at 25 C and 15"A, relative humidity. Cultures were transferred to a modified Tullgren appara- tus. Live mites were forced to pass through six layers of cheese cloth to escape the heat produced by a 60 W light bulb. Mite bodies were frozen and subsequently defatted with ether, extracted overnight in 100 niM ammonium bicarbonate and centrifuged at 12 000 g. The supernatant was dialysed against distilled water and frozen at - 2 0 C until use. Extract made from isolated D. pteronyssinus bodies was kindly provided by Shirley Williams, Hollister Stier. Spokane. WA. Total IgE and specific IgE antibodies Total serum IgE was measured by two-site radioimmunoassay (RIA) [4.20]. Briefly, removawell plates were coated overnight with monoclonal antibodies (MoAbs) to heavy chain determinants on IgE (CIA/E/7.12 and CIA/E/4.15. kindly provided by Dr Andrew Saxon. University of California. Los Angeles. CA). Dilutions of a control serum or patients' sera were added, followed by '-'^I-labelled goat anti-human IgE. Anti-human IgE was prepared by immunization of a goat with purified Fc fragment of the IgE PS myeloma protein in Freund's complete adjuvant and subsequent affinity purification of the antibodies over a PS Fc epsilon fragment immunosorbent [21]. IgE results were expressed as international units/ml (I lU/mi, about 2 4 ng/ml of IgE). Specific IgE antibodies to D,pteronyssinus. D. farinae. cat epithelium. German cockroach {Blattella germanica). A.spergittus fumigatus. short ragweed pollen and rye grass pollen were measured by RAST [4]. In order to decrease background binding on RAST. horse serum was used as diluent in all steps of the assay. Extractsoffi. »-o/>/(a//.v mite bodies and whole B. tropicalis culture were used to coat CNBractivated RAST discs (lO/ig protein/disc) for measuring IgE antibody to B. tropicalis. Preliminary experiments with four sera showed that optimal binding was seen with 10 /ig B. tropicalis protein/disc. All assays for IgE antibodies were quantitated on the basis of a control curve, using D. farinae discs and serial two-lbld dilutions of a mite allergic serum pool (UVA 87/01) that had been substandardized against an International Reference Serum Pool (NIBSC Code no. 82/528). The UVA 87/Oi control serum pool contains 1000 RAST U/ml of IgE antibody to D. farinae. One unit of IgE antibody in the RAST is approximately equivalent to 01 ng of IgE [4]. Allergen levels in hou.se dust Samples of house dust were obtained from four sites of each patient's house: bedding, bedroom floor. TV room and kitchen, using a hand-held vacuum cleaner (Electrotux. Sao Paulo, Brazil). Floor dust was collected by vacuuming an area of 1 n r for 2 min [22]. Kitchen samples Dust mite allergens among children in Brazil included cabinet and floor dust. Fine dusl was obtained by sieving through a 0-3-mm mesh screen. Samples of 100 mg were extracted overnight in 4 C in 2 ml boratebuffered saline (BBS), pH 8-0. After centrifugation, supernatants were stored at — 20"C prior to immunoassay. Group I Dermatophagoides allergens {Derp I and Der / I ) , cat allergen Fel d I, and cockroach (B. germanica) allergen Bla g I were measured by two-site MoAb-based ELISA [23-25]. Group II Dermatophagoides sp. allergens were quantified by MoAb-based RIA [26]. Reference standards containing known levels of each allergen were used to make control curves and the results were expressed as /xg/g of dust or ng/ml of extract. Cockroach assays were quantitated based on a reference German cockroach extract (LJVA 89/01), and results were expressed as arbitrary units of Bla g I/g of dust [25]. Immunoabsorption studies Absorption experiments were carried out as described by Heymann et al. [27]. CNBr-activated Sephurose was coupled with 10 mg D. pterony.ssinus whole body extracl/g ofgeKorwith lOmgfi. //•(j/7/c«//.vbodyextract/gofgel,or with 10 mg human scrum albumin (HSA)/gof gel. It was estimated that 12 mg Der p I/g of gel bound to the Sepharose (99'>(> of Der p I added). Sera from six Brazilian children (12 ml diluted 1/2 or 1/8) who had > 200 U of IgE antibody to both D. pteronyssinus and B. tropicalis were absorbed with 0-25 mi AO'Vo Sepharose beads for 6 hr and then separated by centrifugation. Serum from an American patient allergic to D. pterony.ssinus but without measurable IgE antibody to B, tropicalis was also absorbed. Measurements of IgE antibody to D. pteronyssinus and B. tropicalis were carried out by RAST on all samples post-absorption. Absorption results were calculated as the mean of the percentage of reduction in IgE antibody observed with each dilution. Scrum IgG antibody levels to Der p I were measured by antigen-binding RIA [28]. Absorption by mite immunosorbent was compared with absorption by HSA immunosorbent for each sample. Results IgE response to inhalant allergens Total serum IgE levels were >400 IU/ml in 18 out of 20 children (geometric mean <gm) 1092 IU/ml. range 967031). In each scrum the highest levels of IgE antibodies were to mite antigens; IgE antibodies to D. pteronyssinus were > 200 RAST U/ml in 19 out of 20 children (Fig. I). The levels of IgE antibodies to D.farinae were on average 2-3-fold lower; however, there was a close correlation 435 10 000 - 1000 - 100 10 ai n>17 Ox 17 Ox 15 Ox 15 B-r Dpi Dt Cai Cl Rye rw Fig. 1. Levels of serum IgE antibodies to A. fumi^atu,s (AO. B. tropicatis (Bt). D. pteronyssinus (Dpt), D. farimie (DO, cat, cockroach (cr), rye grass and ragweed (rw) pollens in 20 miteailergic asthmatic chiidren from Sao Paulo. Values shown below the lower dashed line (for example. Ox 15 for A. fumigatu.';} represent undeiectable IgE antibodies ( < 4 D/ml). The upper dashed line represents levels of 200 U/ml of specific IgE antibody (equivalent to about 20 ng of IgE) which we consider as a moderate positive RAST [4]. between the results for the two Dermatophagotdes species (r-0-90, /»< 0-001). Levels of IgE antibodies to B. tropicalis of > 200 RAST U/ml were observed in 11 out of 20 sera or 13 out of 20 sera using the whole mite culture or the mite body B. tropicalis extracts, respectively. A good quantitative correlation was seen between the results for the two B. mj/j/ca//.s'extracts (/• = 0-80. f <0-05). Levels of IgE antibodies to D. pteronys.sinus{gm 1585 RAST U/ml) were significantly higher than the levels of B. tropicalis (gm 358 RAST U/ml) (/-3-78, /'<0-001, Student's ttest). RAST analysis using B. tropicalis food medium (10 ^g protein/disc) showed < 1% binding, as compared to up to 31"/) binding on B. tropicalis discs. IgE antibodies to B. tropicalis in a group of nine patients from Virginia. U.S.A., allergic to D. pteronyssinus 045-10000 RAST U/ ml) were very low and in no case greater than 5% the results obtained in the D. pterony.ssinus RAST (data not shown). IgE antibodies to cat. cockroach. A. fumigatus, ragweed and rye grass were <80 RAST U/ml (Fig. 1). Exposure to indoor allergens Mite, cat and cockroach allergens were measured in 79 dust samples from the 20 houses visited (one kitchen dust sample was not available). The highest levels of Der p I and group II allergens were found in bedding samples 436 L. Karla Arruda et a!. The specificity of IgE antibodies to £>. pteronyssinus and B. tropicalis was compared by immunoabsorption using six selected sera from Brazilian patients with asthma and RAST > 200 U/ml to both mite species. M ost IgE antibodies to D. pteronvssinus were removed by absorption of the sera with D. pteronyssinus extract (95-8 + 3%), but only 32% of the IgE antibodies to D. pteronyssinus were absorbed with B. tropicalis extract {Fig. 3a). Similarly, most IgE antibodies to B. tropicalis were absorbed with B. tropicalis extract (95-1 ±3-9%), but only 36% ofthe IgE antibodies to B. tropicatis were removed following absorption with D. pterony.ssinus extract (Fig. 3b). Absorption of sera with B, tropicalis had no effect on levels of IgG antibodies to Der p I, while absorption with D. pteronyssinus removed >95"A> of IgGanti Der p I from all sera (Fig. 3c). 500 r 50 a A - LT. 0-5 |2/ig/g 0-1 0-1 0-5 I lO^g/g 5 50 500 Group It mite allergen Fig. 2. Measurements of Der p I and Group II allergens, expressed in /ig/g of fine dusl., in 79 samples of house dust, collected from four sites of eaeh patienl's home: bedding (•), bedroom floor (A). TV room {•) and kilehen (O). Levels of 2/ig/g and 10 /jg/g of dusl. which have been proposed as risk faelors for sensitization and development of acute asthma in mite-allergic individuals, rcspeetively, are indicated as dashed lines. (gm 38 4 /tg/g and 36-6 ;(g/g, respectively), followed by bedroom floor (gm 5-7 /ig/g and 5-6 /(g/g), TV room (gm 4-1 ^g/g and 5-6 /ig/g) and kitchen (gm 0-5 /ig/g and 0 9 /ig/g) (Fig. 2). There was an excellent correlation between levels o^ Der p I and group II allergens (/• = 0-94, / ' < 0 0 0 l ) . Derfl was undetectable or found in very low levels ( < 0-5 //g/g). Levels ofcat allergen Eel t/1 > 8 /tg/g of dust (a level which has been consistently associated with presence of cat(s) in the house) were found in two houses. Cockroach allergen was detected in five houses; however, the levels were < 10 U/g in each case. Anaty.s'is of cro.ss-reactivity between D. pteronyssinus and B. tropicalis Extracts of D. pteronyssinus and D. farinae prepared in Brazil had Group I and Group II allergen levels ranging from 4-7 to 9-3 /ig/ml. Extracts of B. tropicatis. prepared from two different cultures, had undetectiible or < 50 ng/ ml Dermatophagoides sp. allergens. The absence of Group 1 allergens in B. tropicalis was confirmed by both monoclonal and poiyclonal antibody-based immunoassays (Table I) [29]. These results suggested that B. tropicalis produced allergens that were distinct from the major D. pteronyssinus allergens. Discussion The children in our study were all exposed to levels of Der pi >2/ig/g of dtist, and levels > 10/ig/g of/)f/-/> 1 were detected in 18 out of 20 ofthe houses. Those levels have been proposed as risk factors for sensitization and development of acute attacks of asthma, respectively [6]. The mean levels ofmite allergens in Brazilian houses were higher than levels reported in similar studies performed in the U.S.A [22] and in England [5]. These high levels probably reflect the fact that the relative humidity in Sao Paulo is 70-80% year round, with temperatures of 2025' C,i.e. favorable conditions for mite growth. The Derp l:Group I! ratio in our dust samples was 0 96:1. Sakagushi et at. [30] have reported a similar ratio (0 8:1) in Japanese floor dust samples. Levels ofcat allergen were undetectable in most houses: only one out of 20 patients reported the presence of cats in the house. Our results suggest that cockroach may not be as signilicunl a cause of sensitization in Sao Paulo as house dust mites. However, the population we have studied was primarily selected for mite sensitivity, and it is possible that cockroach allergy has a role in asthma among a different group of patients. Although B. germanica has a cosmopolitan distribution, the cockroach fauna in Sao Paulo may include other species, which do not produce antigen detected by the assay for Bla g I. The Brazilian children with asthma had high levels of specific IgE antibodies to mites in their sera. We have presented the results for D. pterony.ssinus IgE antibodies and B. tropicalis IgB antibodies as if the RAST units for each were equivalent. The comparison of RAST units for different allergens assumes that the preparations of the discs is optimal for each allergen. We tested different extracts and concentrations of B. tropicatis in order to optimize the RAST, and believe that the RAST values Dust mite allergens among children in Brazil 4M Table L Group I and Group II allergen levels in mite extracts Der p I Dcrf\ Group I* Group II (ng/m!) (ng/ml) (ng/m!) (ng/m!) D. pteronyssinus Sao Paulo. Brazil Sao Paulo, Brazil D. farinae Blomia fropicatis Sao Paulo. Brazil Sao Paulo. Brazil Food medium 6580 I58t < 10 <10 <10 4730 <10 <IO 8875 5125 <50 <50 3800 9300 20 <!0 Blomici tropicatis Tampa, FL <IO <IO <50 <!0 Mite extracts Sou ree * Assayed by inhibition RIA using affinity purified rabbit antibodies to crossreacting determinants on Der p 1 and DerfX [29]. t Positive results for the D, farinae extract in the Der p I assay reflect weak crossreactivity in the MoAb assay for Der p I, which has previously been estimated at 1-2'M, [23]. 10 000 lOOOh 100 - HSA Dpt HSA Bt HSA Dp) HSA Bt S HSA Dpt HSA Bl Antigen used Fig. 3. Sera from six selected Brazilian asthmatic children allergic to both D. pteronys.tinus (Dpt) and B. tropicalis (Bt) (•). and from one American asthmatic patient with IgE antibodies to D. pteronyssinus but not lo B. tropicalis (O) were absorbed with either D. pleronyssinus. B. rropicalis or human serum albumin (HSA) Sepharose. Comparisons of IgE antibody levels to cither D. pteronyssinus (a) or B. tropicalis (b) foHowing absorption with these antigens arc shown, (e). Effect of immunoabsorption on IgG antibodies to Dcr p I, as determined by antigen-binding RIA [28]. obtained are broadly comparable with those of D. pterony.ssinus. Positive results observed iti the D. farinae RAST were presutnably due to the fact thai IgE atitibody to antigens of either Dermatophagoides species are highly cross-reactive in humans. Rye-grass- and ragweed-speciHe IgE antibodies were not found in these sera, probably because of lack of exposure. With the exception of the Southern provinces, no grass pollens of allergenic importance have been reported in Brazil [31]. Children from Third World countries are exposed to intestinal parasites (helminths) which can be potent stimuli for inducing marked elevation of IgE and can potentiate the production of IgE against non-parasitic antigens [32]. Although the specificity of the potentiated IgE has not been established, there is good evidence that it is not directed against inhalant allergens [32]. Thus, it seems unlikely that the high levels of IgE to mites we see in our patients are a cotisequence of parasitic infection. High levels of total IgE may increase background binding on RAST; however, the low results obtained on rye grass and ragweed RAST do not support this hypothesis. The total IgE levels found in sera of otir children were at least five-fold higher than mean levels reported in two separate studies on epidemiology of acute asthma in adults, carried out in Charlottesville. Virginia [4] and in Wilmington, Delaware [33]. These results suggest that some of the children may have had parasitic infection. In future studies, it would be prudent to examine for parasitic infections and to investigate whether parasites infiuence [gE antibody responses in urban populations of BraziUati children. Previous studies performed in Sao Paulo., based on mite counts and microscopic identification of mites, have shown D. pteronyssinus and B. tropicalis to be the most prevalent mite species in housedust. In addition, seasonal variation of both mite species has been reported, with peaks of frequency in March April and in AugustNovember in house dust [7]. The present study did not focus on counting mites in dust; however, analysis of house dust samples has shown that D. pteronyssinus represented > 50"At of the mite species in all but one house; B. tropicalis was identified in dust from four of the houses, representing 5-26"A> of the mites present. These numbers appeared to be lower than has been reported previously. Other mites identified included Euroglyphus maynei and Cheyletus sp.., and no species of D. farinae were found. 438 L. Karla Arruda et al. Absorption experiments revealed that most of the IgE antibodies to B. tropicalis were directed against speciesspecific antigens, however a smaller proportion recognized antigens shared with D. pteronyssinus. Since we found undetectable or very low levels of Der p I, Der fl or Group II Dermatophagoides allergens in two different B. tropicalis extracts, it seems likely that cross reactivity is associated with other allergens [34]. Van Hage-Hamsten et al. [35] have recently reported that B. kulagini is another important Blomia species in Brazil: however, no comparative studies on prevalence or on the frequency of sensitization to both species have been performed. Their RAST inhibition results using sera from Swedish farmers or allergic subjects from Brazil also suggested that D. pteronyssinus and B. kulagini have predominantly speciesspecific allergens [35]. 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