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"Residu protein pada barang jadi lateks alam (Heuea brasiliensis) yang banyak dipergunakan untuk perlengkapan kedokteran dan rumah tangga (sarung tangan, catether, tensimeter, kondom, karet busa dll) dilaporkan dapat menyebabkan reaksi hipersensitivitas tipe I (tipe cepat) yang dapat berakibat fatal bagi pemakainya sehingga dikhawatirkan konsumsi lateks alam di pasaran dunia akan menurun dan beralih menggunakan lateks sintetis yang lebih aman. Untuk memberikan jaminan keamanan produk lateks, Food and Drug Administration (FDA), Amerika Serikat dalam websitenya http:/www.fda.gov /cdrh/manual/glovmanl.pdf akan menerapkan labeling rendah protein allergen (hypo allergenic protein) dengan persyaratan yang ketat.Indonesia sebagai market leader lateks alam setelah Thailand perlu mengantisipasi dampak labeling FDA dan reaksi konsumen internasional melalui serangkaian langkah-langkah nyata. Oleh karena kadarnya sangat rendah dan jenis proteinnya khas, maka teknik deteksi protein allergen yang spesifik dan sensitive. Perangkat imunodiagnostik merupakan teknik yang paling sesuai dari segi kepekaan, kespesifikan dan kemudahan penerapannya di lapang untuk deteksi protein allergen. Perangkat deteksi protein allergen yang spesifik, sensitive, mudah dan mudah belum tersedia di pasar. Penelitian ini bertujuan untuk mengetahui pola pengikatan protein allergen lateks dengan IgE individu sensitive dan merakit perangkat diagnostik imunologi menggunakan antibody anti protein allergen yang sesuai. Penelitian Tahun I mencakup kegiatan penelitian lapang (uji reaksi sensitisasi allergen pada kelompok terpajan dan tidak terpajan secara cross sectional dan uji skin prick test) dan laboratorium meliputi koleksi IgE serum manusia peka, penetapan kadar protein berbagai produk lateks, deteksi protein allergen dengan dot blot menggunakan IgG kelinci antilutoid, deteksi protein allergen dengan IgE manusia peka dengan teknik ELISA dan dot blot, identifikasi protein allergen dengan SDS-PAGE dan Western blotting menggunakan IgE manusia peka dan IgG kelinci antilutoid serta elusi protein allergen dari preparative gel SDS-PAGE dengan elektroeluter untuk sumber antigen terpilih untuk perakitan antibody monoclonal yang akan dilakukan pada Penelitian Tahun II. Hasil penelitian menunjukkan bahwa uji prevalensi sensitisasi lateks telah dilakukan pada karyawan industri lateks PT Mitra Rajawali Banjaran (MRB) dan PT Sugih Instrumendo Abadi (SIA) Padalarang Jawa Barat seperti ditunjukkan pada hasil skin prick test positif pada karyawan PT Mitra Rajawali Banjaran yaitu sebesar 1 8 % (n = 24) dan PT SIA sebanyak 25 % (n = 200). Skin prick test positif juga dijumpai pada perawat dan peneliti laboratorium. Hasil skin prick test bervariasi antara positif satu (+) hingga positif tiga (+++) dan dijumpai baik pada karyawan yang langsung berhubungan dengan bahan dan produk lateks maupun yang tidak langsung berhubungan dengan produk (karyawan bagian administrasi, umum, satpam dan pemasaran). Teknik ELISA sandwich lebih sensitive ibanding ELISA tak-langsung dan ELISA langsung untuk mendeteksi keberadaan protein allergen lateks. Dengan teknik ELISA sandwich dapat diketahui bahwa kandungan protein allergen pada kondom (0,085-0175 OD ELISA) lebih rendah disbanding tensimeter (OD ELISA 0,088-0,693). Tensimeter yang dibuat dengan teknik iradiasi mengandung protein allergen lebih rendah disbanding tanpa iradiasi baik pada bagian bulb maupun lengan. Kandungan protein allergen bulb dan lengan tensimeter iradiasi yaitu 0,088 dan 0,184 dan pada tensimeter tanpa iradiasi yaitu 0,234-0,693 dan 0,264-0,283 OD ELISA. Protein allergen pada fraksi lutoid dan serum-C dalam lateks dapat dideteksi secara kualitatif dengan uji dot blot baik dengan IgE serum manusia peka dan IgG kelinci antilutoid. Dot blot dengan IgG keleinci antilutoid memberikan sinyal yang lebih kuat dibanding IgE serum manusia peka. Protein allergen pada lutoid dan serum-C lateks dapat diidentifikasi dengan Western blotting. Protein allergen yang teridentifikasi dalam lutoid yaitu memiliki BM 10, 14,5 21, 23, 32, dan 36 kDa. Protein allergen dalam serum C memiliki BM 17 kDa seta beberapa pita minor pada BM antara 25 kDa - 50 kDa. Sedangkan hasil Westen blotting dengan serum IgG kelinci anti lutoid diperoleh protein alergen yang relatif identik dengan serum IgE hanya intensitasnya lebih kuat. Protein alergen yang terdeteksi dengan IgG kelinci anti lutoid yaitu protein dari yang paling dominan yaitu BM 36, 50, 20, 60, 66 dan 16 kDa dari lutoid Berta BM 20 dan 16 kDa dari serum C. Protein dengan BM 21, 23, 32, dan 36 kD dalam lutoid lateks telah dielusi dari gel SDS-PAGE preparative dan akan digunakan untuk sumber antigen untuk pembuatan antibody pol iklonal dan monoclonal pada Tahun II. Selain pengembangan perangkat deteksi, juga telah dilakukan usaha deproteinasi protein allergen dengan protease basa dari isolat bakteri P6a asal lateks. Protein terlarut pada sarung tangan yang semula kadamya 1836 .iglg dengan penambahan enzim protease kasar 10% yang diaplikasikan bersama surfaktan 1% menurunkan sebesar 73% (500 Rig). Hasil penelitian yang diperoleh pada Tahun I sangat prospektif dan memilki peluang keberhasilan yang cukup besar untuk dapat merakit perangkat imunodiagnostik yang bemilai komersil pada akhir Tahun II karena (1) jenis-jenis protein asal lateks yang diduga kuat bersifat allergen telah diisolasi dan dimurnikan dari preparative gel SDS-PAGE, (2) determinasi individu yang positif terhadap allergen lateks, (3) Vasil pendahuluan kemampuan enzim protease yang diproduksi sendiri untuk penurunan kadar protein sarong tangan, (4) optimasi berbagai teknik immunoassay seperti ELISA, Dot blot dan Western blot. Antibodi poliklonal dan monoclonal spesfik terhadap protein allergen dan teknik deteksinya akan didaftarkan untuk paten sederhana."

"Penelitian ini adalah penelitian berbasis komuniias untuk memperoleh gambaran kadar alergen tungau debu rumah (TDR) dan sensitisasi alergen sebagai faktor risiko asma. Penelitian ini merupakan studi potong lintang dengan responden 3.840 pelajar sekolah menengah pertama dari 19 sekolah yang dipilih secara acak dari 131 sekolah di Jakarta Pusat. Seinua responden mengisi kuesioner International Study on Asthma and Allergy in Children (ISAAC). Dari 3.840 responden, 288 (7,5%) digolongkan dalatn kelompok asma karena mempunyai riwayat mengi selama 12 bulan terakhir. Pada kelompok asma, 207 responden melakukan uji kulit dan 135 diambil sampel debu rumah. Dari 2.601 responden yang tidak memiliki riwayat asma atau atopi lain, dipilih secara acak 274 orang untuk uji kulit dan 165 di antaranya diambil sampel debu rumah. Kadar alergen TDR tidak berbeda bermakna antara kelompok asma maupun non-asma baik untuk Der pi, Der fl, rnaupun Grup I. Risiko asma terutama disebabkan oleh sensitisasi alergen TDR D.pteronyssinuss (rasio odds (OR):12,68, interval kepercayaan (Cl): 7,50-21,44), D.farinae (OR: 10,50, CI: 6,35-17,34), serta kecoak (OR-.5.57, Cf:3,44-9.0l). Perbedaan risiko asma tidak berhubungan dengan tinggi rendahnya kadar alergen TDR tetapi oleh sensitisasinya. Kesimpulannya, sensitisasi alergen TDR dan kecoak perlu diperhatikan dalam upaya menurunkan risiko dan prevalensi asma. (MedJ Indones 2006; 15:55-9).

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This study is a community-based study to get an overview about House Dust Mite (HDM) allergen level, allergen sensitization as risk factors of asthma. This is a cross-sectional study on 3,840 students from 19 junior high schools, aged 13-14 years. AH of the respondents filled out the International Study on Asthma and Allergy in Children (ISAAC) questionnaire. Of 3840 respondents, 288 (7.5%) were assigned to asthma group (experience wheezing during the last 12 months). The skin prick test was performed on 207 respondents and the house dust mite was collected from 135 respondents. Of 2601 respondents in non-asthma group, the skin prick test was randomly performed on 274 respondents and the house dust mite was collected from 165 respondents. There is no significant difference on HDM allergen concentration for Der pi, Derfl or Group I between asthma and control group. The risk of asthma was caused especially by sen D.pteronyssinus (crude odds ration (OR): 12.68, 95% confidence interval (Cl): 7.50-21.44), D.farinae (OR: 10.50, Cl: 6.35-17.34) and cockroach allergen {OR: 5.57, Cl:3.44-9.01). The risk for asthma was not correlated with the level of house dust mile allergens but its sensitization. In conclusion, sensitization to HDM and cockroach allergen should be concerned in order to reduce risk and prevalence of asthma. (MedJ Indones 2006; 15:55-9)."

"General aim: this study aims to determine the prevalence of sensitization reaction on exposed and unexposed groups of workers at a latex factory. Specific aim: to determine the latex allergen sensitization reaction and factors such as atopy, age, sex, smoking, length of working, exposure concentration.Sample and method: the sample population consists of workers at a sphygmomanometer and condom factory who were, exposed and un-exposed to latex material at the factory. The number of sample was determined based on the two proportion formula.This study was a cross-sectional study using the Chi square test, Fisher test, Mann Whitney test, and Kruskal Wallis test.The results of the study and conclusion: The prevalence oj latex allergen sensitization reaction in the exposed group was 2.38%, while in the un-exposed group 3.33%. The correlation between exposed and un-exposed group to the latex allergen sensitization reaction (overall) was not significant (p=0.l8I), but if the correlation of each factor was calculated, the most significant correlation was between the negative latex allergen sensitization reaction with a strength of +2 or above (p=O.OJ4).In this study, atopy (p=0.000), exposed concentration (p=0.014), and smoking (p=0.018) are factors that were associated with latex allergen sensitization reaction. Workers at the condom factory were at higher risk than those at the sphygmomanometer factory, with a prevalence oj'7.14% and 2.31% respectively."

"Background: nowadays, specific IgE measurement has been conducted in Indonesia, however there is still lack of data regarding diagnostic test to detect inhalant allergen in patients with respiratory allergies.This study aimed to determine the accuracy of specific IgE test in diagnosing specific sensitization of inhalant allergen in patients with respiratory allergies.Methods: this was a cross sectional study in patients with respiratory allergies and part of epidemiology study regarding to specific IgE sensitization in Allergy-Immunollogy Division, Cipto Mangunkusumo Hospital, Jakarta within November-December 2016. Measurement of specific IgE sensitization using Immunoblot method (Euroline, Euroimmun AG, Germany). The tested allergen is house dust mites Dermatophagoides pteronyssinus (Der p), Dermatophagoides farinae (Der f), Blomia tropicalis (Blo t) and cockroach Blatella germanica (Bla g). The result is compared with gold standard, skin prick test. The diagnostic result includes sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (LR+), and negative likelihood ratio (LR-).Results: a total of 101 patients were enrolled; 77 (76.2%) were women. Patients mean age was 38.8 years old. Based on SPT, sensitization was highest for Blo t (76.2%), followed by Der p (70.3%), Der f (69.3%), and Bla g (41.6%). Specific IgE-sensitization was highest for Der f (52.9%), followed by Der p (38.2%), Blo t (33.3%) and Bla g (10.8%). Der p allergen had 50.7% sentivity, 90% specificity, 92.3% PPV, 43.5% NPV, 5.1 LR+ and 0.1LR-. Der f showed 71.4% sensitivity, 87.1% specificity, 82.6% PPV, 57.4% NPV, 5.5 LR+ and 0.3 LR-. Blo t allergen had 41.6% sensitivity, 91.7% specificity, 94.1% PPV, 32.8% NPV, 5.0 LR+, and 0.6 LR-. Bla g allergen had 23.8% sensitivity, 98.3% specificity, 90.9% PPV, 64.4% NPV, 14.5 LR+ and 0.8 LR-.Conclusion: serum specific IgE testing to common inhalant allergen in patients with respiratory allergy showed only low-to-moderate sensitivity, but high specificity and PPV. This new assay can be used to diagnose allergen sensitization in the population with high prevalence of TDR and cockroach."

"AbstrakPURPOSE:House dust mites (HDM) are major allergens that cause allergic rhinitis (AR). Allergen-specific subcutaneous immunotherapy (SCIT) has been shown to be clinically beneficial in many clinical trials. Such trials, however, are not reflective of all patient populations. The aim of this study was to describe the efficacy and safety of SCIT in routine clinical practice in Korean adults with AR sensitized to HDM.METHODS:We reviewed medical records of 304 patients with AR treated at an allergy clinic of a tertiary hospital using SCIT with aluminum hydroxide-adsorbed allergen extract targeting HDM alone or with pollens for at least 1 year from 2000 to 2012. Patients with asthma were excluded. Rates of remission, defined as no further requirement of maintenance medication, over time were determined by means of life tables and extension of survival analysis. Specific immunoglobulin E (IgE) levels to HDM were categorized into 6 classes.RESULTS:The mean time until achieving remission was 4.9±0.1 years, and the cumulative incidence of remission from AR was 76.6%. Severe AR (odds ratio [OR], 0.40; 95% confidence interval [CI], 0.23-0.69; P=0.001), specific IgE levels to HDM ≥17.5 kU/L (OR, 1.85; 95% CI, 1.01-3.37; P=0.045), and duration of immunotherapy ≥3 years (OR, 7.37; 95% CI, 3.50-15.51; P<0.001) were identified as significant predictors of clinical remission during SCIT for patients with AR sensitized to HDM. Overall, 73 patients (24.0%) experienced adverse reactions to SCIT, and only 1 case of anaphylaxis (0.3%) developed.CONCLUSIONS:SCIT with HDM was found to be effective and safe for patients with AR. Specific IgE levels to HDM and a duration of SCIT ≥3 years may be predictors of clinical responses to SCIT in AR patients."

"House dust mites (HDM) are an important inhalant allergen in allergic asthma. However, molecular diagnostic study of specific IgE to HDM allergens has not been done in Indonesia. in addition, the association of quantitative specific IgE measurement with asthma severity has not been investigated. This study aimed to investigate difference of serum quantitative specific IgE levels induced by Dermatophagoides (D)."

"ABSTRAKLatar Belakang: Atopi yang ditandai dengan sensitisasi (produksi IgE) terhadap alergen merupakan faktor risiko untuk terjadinya penyakit alergi. Karena komposisi genetik cenderung stabil, peningkatan prevalensi penyakit alergi diduga disebabkan oleh faktor lingkungan yang berubah.Tujuan: Mengetahui faktor yang memengaruhi sensitisasi terhadap alergen hirup dan munculnya manifestasi penyakit alergi pada populasi dewasa muda di Indonesia.Metode: Penelitian potong lintang dilakukan pada mahasiswa baru Universitas Indonesia tahun 2019. Mahasiswa diminta mengisi kuesioner berisi data demografi, kuesioner International Study of Asthma and Allergies in Childhood (ISAAC), menjalani pemeriksaan fisik dan uji cukil kulit terhadap lima alergen hirup (kecoa, Dermatophagoides farinae, Dermatophagoides pteronyssinus, bulu kucing, epitel anjing). Sensitisasi ditandai dengan terdapat setidaknya satu hasil positif pada uji cukil kulit. Jika terdapat hasil positif pada dua atau lebih alergen, subyek disebut mengalami polisensitisasi. Manifestasi alergi yang dinilai berupa asma, rinitis alergi, dan dermatitis atopi.Hasil: Proporsi sensitisasi adalah 44,8% (128 dari 286 subyek), sedangkan proporsi manifestasi penyakit alergi adalah 57,7% (165 dari 258 subyek). Manifestasi penyakit alergi didapatkan pada 84 (65,6%) subyek dari subkelompok yang tersensitisasi. Sensitisasi ditemukan lebih banyak pada laki-laki (OR 2,25; IK95% 1,38-3,71; p=0,001) dan subyek yang lahir secara caesar (OR 2,46; IK95% 1,22-5,06; p=0,013), sebaliknya lebih sedikit pada subyek yang berasal dari urban (OR 0,54; IK95% 0,32-0,90; p=0,019). Subyek yang tersensitisasi cenderung untuk memiliki manifestasi penyakit alergi (OR 1,79; IK95% 1,10-2,95; p=0,020). Pada subkelompok yang tersensitisasi, manifestasi penyakit alergi ditemukan lebih banyak pada subyek yang tinggal di urban (OR 2,58; IK95% 1,15-6,01; p=0,024), obese (OR 5,25; IK95% 1,35-34,92; p=0,036), dan mengalami polisensitisasi (OR 2,26; IK95% 1,01-5,10; p=0,046).Simpulan: Sensitisasi terhadap alergen hirup dipengaruhi oleh jenis kelamin lakilaki, status urban, dan riwayat persalinan caesar. Munculnya manifestasi penyakit alergi dipengaruhi oleh adanya sensitisasi. Pada subkelompok yang tersensitisasi, munculnya manifestasi penyakit alergi dipengaruhi oleh status urban, obesitas, dan polisensitisasi.

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ABSTRACTBackground: Atopy marked by allergen sensitization (IgE production) is a risk factor for allergic diseases. Since genetic composition tends to be stable, the increase of allergic diseases prevalence is suspected due to changing environment factors.Purpose: To identify the factors affecting sensitization to inhalant allergen and allergic diseases manifestation in Indonesian young adults.Methods: Cross-sectional study done on Universitas Indonesia 2019 new students. Students were asked to fill in a demographic questionnaire, an International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire, undergo physical examination and skin prick tests for five inhalant allergens (cockroach, Dermatophagoides farinae, Dermatophagoides pteronyssinus, cat hair, dog epithelium). Sensitization was marked by at least one positive result on the skin prick test. If there were two or more positive allergen results, subject was deemed as being polysensitized. Evaluated allergic manifestations were asthma, allergic rhinitis, dan atopic dermatitis.Result: Sensitization was found in 44.8% (128 out of 286 subjects), while allergic diseases clinical manifestation was found in 57.7% (165 out of 258 subjects). The manifestation was found in 84 (65.6%) subjects from the sensitized subgroup. Sensitization was found more on male (OR 2.25; 95%CI 1.38-3.71; p=0.001) and subjects born by caesarean section (OR 2.46; 95%CI 1.22-5.06; p=0.013), whereas fewer on subjects from urban (OR 0.54; 95%CI 0.32-0.90; p=0.019). Sensitized subjects tended to demonstrate allergic diseases manifestation (OR 1.79; 95%CI 1.10-2.95; p=0.020). In the sensitized subgroup, allergic diseases manifestation was found more on subjects living in urban (OR 2.58; 95%CI 1.15-6.01; p=0.024), are obese (OR 5.25; 95%CI 1.35-34.92; p=0.036), and are polysensitized (OR 2.26;95%CI 1.01-5.10; p=0.046).Conclusion: Sensitization to inhalant allergens was affected by male sex, urbanstatus, and caesarean section birth. Allergic diseases manifestation was affected by presence of sensitization. In the sensitized subgroup, allergic diseases manifestation was affected by urban status, obesity, and polysensitization."

"Latar belakang: Tungau debu rumah TDR merupakan alergen hirup yang penting pada asma alergik. Namun, penelitian diagnostik molekuler menggunakan Imunoglobulin E IgE spesifik akibat sensitisasi alergen TDR dihubungkan dengan derajat keparahan asma alergik belum pernah dilakukan di Indonesia.Tujuan: Mengetahui perbedaan kadar IgE spesifik serum kuantitatif akibat sensitisasi alergen Dermatophagoides D. pteronyssinus, D. farinae dan Blomia B. tropicalis pada asma alergik intermiten dan persisten.Metode: Desain penelitian potong lintang pada pasien asma alergik dewasa yang diundang untuk pemeriksaan IgE spesifik serum dan merupakan bagian dari penelitian payung di Divisi Alergi dan Imunologi Klinik, RS Cipto Mangunkusumo. Derajat keparahan asma ditentukan berdasarkan kriteria Global Initiative on Asthma GINA 2015 dan dikelompokkan menjadi intermiten dan persisten. Pemeriksaan IgE spesifik serum kuantitatif menggunakan metode multiple allergosorbent test Polycheck Allergy, Biocheck GmbH, Munster, Germany . Alergen TDR yang diperiksa adalah D. pteronyssinus, D. farinae, dan B. tropicalis. Perbedaan antara dua kelompok dianalisis dengan uji Mann-Whitney.Hasil: Sebanyak 87 subyek dilibatkan dalam penelitian ini; 69 79,3 subyek adalah perempuan. Rerata usia pasien adalah 40,2 tahun. Enam puluh tiga 72,4 pasien menderita asma dan rinitis alergik. Sebanyak 58 66,7 pasien asma persisten. Gambaran sensitisasi alergen TDR adalah 62,1 D. farinae; 51,7 D. pteronyssinus dan 48,3 B.tropicalis. Median kadar IgE spesifik secara bermakna lebih tinggi pada asma persisten dibandingkan asma intermiten untuk alergen D. farinae 1,30 vs. 0,0 kU/L; p=0,024 dan B. tropicalis 0,57 vs. 0,0 kU/L; p=0,015 . Kadar IgE spesifik D. pteronyssinus lebih tinggi pada asma persisten dibandingkan intermiten 0,67 vs. 0,00 kU/L; p=0,066.Kesimpulan:Gambaran sensitisasi alergen secara berurutan didapatkan D. farinae 62,1, D. pteronyssinus 51,7 dan B. tropicalis 48,3 . Kadar IgE spesifik akibat sensitisasi D. farinae dan B. tropicalis lebih tinggi secara bermakna pada pasien asma persisten dibandingkan asma intermiten. Kadar IgE spesifik akibat sensitisasi D. pteronyssinus lebih tinggi pada pasien asma persisten dibandingkan asma intermiten, tetapi secara statistik tidak bermakna.

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Introduction House dust mites HDM are an important inhalant allergen in allergic asthma. However, molecular diagnostic study using specific IgE level induced by HDM allergens associated with asthma severity has not been done in Indonesia.Objective To investigate the difference of serum quantitative specific IgE levels induced by Dermatophagoides D. pteronyssinus, D. farinae and Blomia B. tropicalis sensitization in intermittent and persistent allergic asthma.Method This was a cross sectional study on adult allergic asthma patients who were invited for serum specific IgE testing. This study was a part of a larger research within the Division of Allergy and Immunology, Cipto Mangunkusumo Hospital. Asthma severity was defined based on Global Initiative on Asthma GINA 2015 criteria and were grouped as intermittent or persistent. Quantitative specific IgE testing was done on blood serum using a multiple allergosorbent test Polycheck Allergy, Biocheck GmbH, Munster, Germany . The HDM allergens tested were D. pteronyssinus, D. farinae, and Blomia tropicalis. Difference between two groups were analyze using Mann Whitney test.Results A total of 87 subjects were enrolled in this study 69 79.3 were women. Mean patients rsquo age was 40, 2 years. Sixty three 72.4 patients had asthma and allergic rhinitis. Fifty eight 66.7 patients were classified as persistent asthma. The prevalence of sensitization was 62.1 D. farinae, 51.7 D. pteronyssinus, and 48.3 Blomia tropicalis. The median of specific IgE levels is significantly higher in persistent asthma compares to intermittent asthma induced by D. farinae median 1.30 vs. 0.0 kU L p 0.024 and B. tropicalis median 0.57 vs. 0.0 kU L p 0.015 sensitization. Level of Specific IgE D. pteronyssinus is also to be higher in persistent asthma than the level measured in intermittent asthma 0.67 vs. 0.00 kU L p 0.066.Conclusion Sensitization of HDM allergens is shown to be highest for D. farinae 62.1 , followed by D. pteronyssinus 51, 7 and Blomia tropicalis 48, 3 . Specific IgE level induced by D. farinae and Blomia tropicalis sensitization are significantly higher in patients with persistent compares to intermittent asthma, whereas specific IgE level induced by D. pteronyssinus sensitization to be higher in persistent asthma although not statistically significant."