RESUME JURNAL IMMUNODIFFUSION Nama Yustika Nur Zannah NIM (08061 181823006 Kelas B Dosen Pembimbing _: 1. apt. Rennie Puspa Novita, M. Farm. Klin 2. apt. Vitri Agustiarini, M. Farm DIAGNOSIS OF ASPERGILLOSIS BY DOUBLE IMMUNODIFFUSION METHOD WITH HOME-MADE ASPERG’US AN ANTISERA. IGENS AND LABORATORIUM MIKROBIOLOGI FARMASI JURUSAN FARMASI FAKULTAS MATEMATIKA DAN ILMU PENGETAHUAN ALAM UNIVERSITAS SRIWIJAYA 2021Diagnosis a Aspergillosis Bu Dowlte (ammumodipeusion arrettred itt, Home amracle Asperayllas Arctineng ond Auctcrewn Bertut Seperailledis onan rong pera adelaty fap thats pada, Sistem perm apatnn. Remeynlirt dune eicroat tone ofl Mafalert Load Sivflay siden juny ave wade Corriclin Lecey As pevoystlees. Asati ean 4 Ms bat hon dane oridin rhon pb seat parca pela clan clapat Aevhontifiy lato Peenembarrnpes fomageliie tae Sertitevitors Sepunt: uma Phstvins ify olvectrtir ahs ter — Bie, Lorn olera: bvorbiopal morale ceepovegi las Cabpa). 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Ae proses pereing ata aetna “ye ease. bing arroging /bevhan ee ee uae Cae oe ev lef sev pi perhadap Orbe, teveeg tear Anas penquarnn clo Manebies Fecavn Sevagarn fan «ven et hopeDAT TAL RUstaca Rittonn Tyigimngig at, Angkonn Chaipragert s Such ar cha- Noemratan ats, 199%, Magness OF Uperoyiliogs Dotste Lmmunod feurion etirorf Loli, eure urteele Asperas lus Antigens and Arnrteva, Medical Journal oF SVinakhoavimaiyot, VelC1) > T-W. FAmudiivaty ones AncunnoR ans ane Diagnosis of aspergillosis by double immunodiffusion method with home-made Aspergillus antigens and antisera Guntunsitaa Dis ovat Nittaya Tripinyipap, M.Sc. (Microbiology)* Angkana_ Chaiprasert, Dr. rer. nat.’ Suchai_Charoenratanakul, M.D., MRCP." Abstract Aspergillosis frequently affects respiratory tract. The definitive diagnosis is difficult. In this study, double immunodiffusion method was developed for detection of precipitating antibodies to Aspergillus species in patients sera by a home-made battery of reagents, composed of culture filtrate antigens and thelr homologous rabbit antisera of A,fumigatus B-1172, Aflavus B-15, A. niger 107, A nidulans B-190 and A. terreus 8-985. Three hundred and fourty nine sera which comprised of 129 from patients with suspected aspergiliosis,30 from patients with lung cancer, 18 from patients with other systemic mycotic infections, 34 from patients with meliodosis, 17 from patients with active pulmonary tuberculosis and 121. from normal individuals were tested. Positive precipitating antibodies by double immunodiffusion method were found in 27.13 % (35/129) of patients with suspected aspergillosis while false positive resuts were found in 1.18 % (4/220) in control groups. unfada = Gas Tasfiniymw, anv. qadosnewy Soann arwusstaSg, — Us.e qéu—wWSrySauna, wv. Aspergilosis aincemurielantweuumnawmels mibinsiteds th naWrzuunidartodonslaunsesindadannefilaeinteelsa ain umislaet! nsevhbidacl@lumeugta — wnsazivlunnsitedeSedosoauioyasney UssnauTumanintelusinaaniaimizuanite ide nrmcieiadnaasen saute SEovianwtundos lunsAnwrnisillaviaw anew uenduefiedannacnow * Department of Microbiology, Faculy of Medicine, Sinakhsrnwiot University anaiangatinu reunnuman’ amvinendurtunbunsilea vaeavfins ** Deparment of Microbiology, Faculy of Medicine, Mahidol Univesiy mainngadsinn acsumnumaniinenena swimndeaan + Deparment of Medicine, Facuty of Meciine, Mahido! University pinorgsedad acucumnuenaadeisremmnina aninmndeninnVolt Not Crignal Arie Medial Jounal of Srnakbaenwizet Wi Aiipradunsdadteluana Aspergiius lufadansiatu serum nei8 double immunodiffusion Tauriaiafem reference reagents Uldtoatuga swisnousae culture filtrate antigens war rabbit antisera flauneinife A. fumigatus 8-1172, A. flavis 8-15, A. niger 107, A. nidulans 8-1390 uae A, terreus 8-985 uaz VWlumsnndeu serum sium 349 diotheasz deldeingioengasiney du uUsenoudie serum vinnduguaeftsedelsn aspergiosis 129 Aaatie, serum sinndaeaniiuseriodon 30 saosin, serum singihedtiulsndadoraeuy aivremelwniadus 18 sande, serum wingilay meloidosis 34 Foe, serum singthefnlsavansse: active 17 dhodw uaz serum ernyaraulsn’ 121 Hod vinnv@nwmuuavanlaeibarenuowiuedfanasnewld 27.13 % (35/129) lwngadilausedelsn aspergiliosis unzwunsuanaas 1.81% (4/220) ‘wings control (wis 1997 ;:1:7-11) Introduction The most common form of aspergillosis is the infection of the respiatory system.” The disease Is caused by inhalation of air bone conidia of Aspergilus species.** The antigens liberated from the conidia will sensitize patients and may contribute to the development of hypersensitivity diseases such as extrinsic asthma, extrinsic alveoltis and allergic bronchopulmonary aspergillosis (ABPA).* Under some circumstances, such as during immunosuppressive therapy or altered host defenses by severe primary "9 the inhaled conidia may germinate diseases, and invade the tissue, resulting in invasive aspergillosis. Saprophytic colonizating of the fungus in pre-existing lung cavities is known ‘as_aspergilloma®*""* or progressive course encroaching upon the lung tissue of indolent cavities will be defined as chronic necrotizing pulmonary aspergillosis (CNPA).*'°"* The non specifi clinical and radiological pulmonary manifestations of aspergilosis create diagnostic difficulty.”"*""* Demonstration of precipitin, antibodies against Aspergilus antigen is widely used’’®" and is very helpful in the diagrosis of 8 ‘asperogillosis."” ** In this study, a battery of home-made reagents according to the COC standards had been prepared for detection of precipitating antibodies in patients sera by double immunodiffusion method. 1, Antigens Aspergillus fumigatus 8-1172 culture filtrate (cr) antigen Aspergilus flavus B-1'5 CF antigen Aspergilus niger 107 CF antigen Aspergilus nidulans B-1390 CF antigan Aspergillus terreus 8-985 CF antigen 2. Antisera Rabbit antiserum of A. fumigatus B-1172 CF antigen Rabbit antiserum of A. flavus B-15 CF antigen Rabbit antiserum of A. niger 107 CF antigen Rabbit antiserum of A. nidulans 8-190 CF antigen Rabbit antiserum of A. terreus 8-985 CF antigenamudiiuariy (2969 Roucumnumaind son FinendTundunduns sm Bis ouuh All Aspergillus species antigens and their homologous antisera were prepared according to the CDC standards and being used as references reagents. 3. Sera 3.1. One hundred and twenty one sera were obtained from normal healthy individuals. 3.2 One hundred and twenty nine sera were obtained from patients with suspected aspergillosis. 3.3. Eighteen sera of other systemic mycotic infections 3.4 Thirty four sera of melioidosis patients 3.5. Thirty sera of patients with lung cancer 3.6 Seventeen sera of patients with active pulmonary tuberculosis All sera were aliquot and kept at-20 °C Until used, Methods Double performed according to Coleman and Kauffman, Immunodiffusion method was Glass slides (1°x3") were coated with thin film of 1 9% purified agar and left dry at room temperature. Three milliliters of molten 1% purified ‘agar was then overlaid on the precoated slide. The gel was allowed to set at room temperature ‘and the wells were punched by a gel puncher, as @ seven-well pattern. The microliters of CF antigen was filled into the central well while the 10 il of either neat rabbit anti-CF homologous or tested sera were added into the peripheral wells. The slide was incubated at room temperature for 24-48 hours in a humid chamber, to allow the precipitation to take place. The slide was then washed with distilled water for 10 minutes at room temperature, and Gipped in 5% sodium citrate for 45 minutes at room temperature to eliminate non specific bands produced by C-reactive protein. Then the slide was again washed with distilled water for another left in normal saline solution overnight at room 10 minutes at room temperature and temperature. The slide was then washed again with distilled water for an additional 10 minutes. The gel was dried under the soaked filter paper With a blow dryer, stained with Coomassie blue ataining solution for 15 minutes and then destained until the background was clear. All of the 349 sera were tested by double Immunodiffusion method against § home-made Aspergillus CF antigens. controls from 5 home-made Aspergillus rabbit The presence of one or more precipitin lines was. in addition, positive antisera were included in every test. indicative of positive results. Results A349 sera obtained from 121 normal individuals, aspergilosis (SUS-ASP), 18 with other systemic ‘mycotic infections (M), 34 with metioidosis (ML), 20 with lung cancer (CA-Lung) and 17 with active pulmonary tuberculosis (TB) were tested with all the five home-made Aspergillus CF antigens.The results are shown in Table 1, which can be seen 129 patients with suspected that the precipitating antibody to A. fumigatus was detected In 31/129 (24.03%) of the patients with suspected aspergillosis while it was not found in the other groups of patients. Small numbers of positive results to A. flavus. A. niger and A. terreus in this suspected aspergillosis group were found. in 1/129 (0.7%), 2/129 (1.55%) and 1-129 (0.77%) respectively The precipitating antibodies could not be detected in all the other group, except the melioidosis group in which precipitating antibodies to A. niger 4/34 could be detected, false positive results were 4/220 or 1.81%.Original Article Volt Not Medi Jura of Sia arnt Group Total Number of sera with posite precipitating antibodies to of subjects No. |A.tumigatus] Atiaws | Aniger | A. nidulans | A terreus 1172 | B15 107 | B-1390 | 8-985 Normal 121 ° ° o ° ° sus-AsP 129) sit 1 2 ° 1 M 18 oO oO o | o oO ML 34 | 0 o o o o cA-tung 30} 0 ° ° o o cy 17| 0 ° ° ° ° Table 1 The number of subjects who gave positive results for precipitating antibodies to the home-made CF antigens of Aspergillus species by double immunodiffusion method. * with slignt cross-reaction with the home-made CF antigens of A. flaws 8-15 Discussion ‘According to the previous studies performed by many investigators it was concluded that the demonstration of specific precipitating antibodies in patients sera with aspergillosis is of value in the diagnosis of pulmonary aspergillosis and can be used together with clinical, cultural and/or histopathologic investigations for the specific of the disease. In this study, the present of specific antibodies to aspergilli assayed by doudle immunodiffusion method will be used as one inclusive criteria for laboratory diagnosis of aspergillosis. Acknowledgement This work was supported by the China Medical Department of Microbiology, Faculty of Medicine, ‘Mahidol University of the laboratory facilities. Board. The author acknowledges the 10 5 sera A.niger107 1 sera References Brouwer J. Dslecton of anibodias against Aspergiusfunigatus ‘campirgon between outa inmunodiusion, LISA anirmunobor alysis It Arch Aleray app nmun 1998 ; 8S : 244-9, Beaumont F. Clie maiettions ot pulmonary Aspergius nections. Mycoses 1988 : 31 Fippon 3. Medial mycology. ed Phiadlphia:W.B. Saunders company, 1988 * 618-50, Campbell ML, Cyten YL Bronchopuimonary asperiisis. A omrdsion ofthe cial and aboatry Kings a 272 patints Invested for bronenopumonay asperosis. 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