{"created":"2023-06-19T07:18:28.005169+00:00","id":3834,"links":{},"metadata":{"_buckets":{"deposit":"68a33caa-6c53-4123-992d-278d30dd1db6"},"_deposit":{"created_by":4,"id":"3834","owners":[4],"pid":{"revision_id":0,"type":"depid","value":"3834"},"status":"published"},"_oai":{"id":"oai:az.repo.nii.ac.jp:00003834","sets":["370:197:376"]},"author_link":["17721","17722"],"item_10006_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2011-12-08"}]},"item_10006_degree_grantor_9":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"麻布大学"}]}]},"item_10006_degree_name_8":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士(学術)"}]},"item_10006_description_22":{"attribute_name":"Abstract","attribute_value_mlt":[{"subitem_description":"At present, no standardized method of clinical mycological examination has been established. Mycological examination in clinical laboratories (e.g., isolation of pathogenic fungi from clinical specimens) is usually performed according to a clinical bacteriological examination method. However, fungi have features that differ from bacteria, such as slow growth rate, which means that the mycological examination must be performed in a different way from that for bacteria.\nSo, the author analyzed mycological examination reports of clinical specimens obtained at the Kitasato University Hospital to investigate the applicability of a clinical mycological examination method. The author wanted to identify the detective rate of fungi that were observed in direct smear preparations, the frequency of the fungi except for Candida spp. and the number of fungal colonies isolated from respiratory specimens, and the incubation days needed to isolate both Aspergillus spp. and Cryptococcus spp. from clinical specimens including dermatophytes from scales.\nFurthermore, using prepared fungal suspensions of Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, the effect for a concentration of the samples by centrifugation, the appropriate inoculum size to isolation medium, and the incubation temperature for isolating the fungi were examined.\n The Clinical and Laboratory Standards Institute (CLSI) suggested a standard susceptibility testing manual for yeast in 1992, and for filamentous fungi in 1998. Since these CLSI manuals were first proposed, improvements to these manuals have continued until now. In Japan, the Japanese Society for Medical Mycology (JSMM) has proposed a susceptibility testing method modified from the CLSI method for yeast and for filamentous fungi.\nA new antifungal agent, Micafungin (MCFG) developed by Astellas Pharmaceutical Co. Ltd., Tokyo in 2002, is a member of the candins and inhibits the synthesis of 1,3-β-D-glucan in fungal cell walls. Although susceptibility testing is necessary for evaluating the clinical effect, the susceptibility testing method of MCFG has not been clarified in these manuals.\nTherefore, the author examined the susceptibility testing method for MCFG according to four manuals of two methods: CLSI M27-A2 (turbidity measurement for yeast) and CLSI M38-A (turbidity measurement for filamentous fungi) from the CLSI method, and JSMM-Y (turbidity measurement for yeast) and JSMM-F (colorimetric method for filamentous fungi) from the JSMM method, by using 48 clinical isolates of Candida spp., 13 clinical isolates of Aspergillus spp., two strains as the quality control (Candida krusei ATCC6258 and Candida parapsilosis ATCC22019), and a reference strain (Aspergillus fumigatus ATCC26430). The author also determined the end point and the inoculum size of the fungi for minimum inhibitory concentration (MIC) measurement.\n\nThe results are summarized below:\n1) Detective rate of fungi from direct smear preparation was 37.0% in 27 deep-seated aspergillosis, and 83.3% in 10 cases of lung or central nervous system cryptococcosis.\n2) Frequencies of fungi, except for Candida spp., isolated from respiratory specimens included Aspergillus fumigatus (62.4%), Aspergillus spp. (18.8%), Cryptococcus neoformas (14.5%), and Cryptococcus spp. (4.3%).\nIn 72.5% of 102 respiratory specimens where Aspergillus spp. were isolated, the number of isolated colonies was one to three, and in only 7.9% of specimens, more than 100 colonies were isolated. Also, in 50% of 14 specimens where Cryptococcus spp. were isolated from lung or bronchial alveolar lavage fluid, the number of isolated colonies was one to three, and in 14.3% of specimens, more than 100 colonies were isolated.\n3) The incubation period needed to isolate all of the 65 isolates of Aspergillus spp. from respiratory specimens was 6 days. In three respiratory specimens isolated Cryptococcus spp., the time required to isolate Cryptococcus spp. was 4 days in one specimen and 5 days in two specimens. In 46 scale samples, the required incubation period to isolate all of 46 dermatophyte isolates was 14 days.\n4) Using a preparation of 10～10^4 cells/ml suspensions for C. albicans, C. neoformans and A. fumigatus, the number of recovered colonies in each suspension with or without centrifugation was compared. In samples containing 10 cells/ml in each suspension, no colonies were detected in the medium without centrifugation; yet some colonies were observed after 15 min centrifugation at 2,000 g.\nUsing a preparation of 10^2 cells/ml in each suspension of C. albicans, C. neoformans and A. fumigatus, the recovered colony size was compared with two incubation conditions: Condition 1, incubation at 35℃ for the first 24 h and at 27℃ for the next 24 h; and Condition 2, incubation at 27℃ for 48 h. C. albicans and A. fumigatus formed larger sized colonies on the plate in Condition 1 than in Condition 2, while C. neoformans formed a larger colony on the plate in Condition 2. In addition, A. fumigatus did not develop any colonies in the first 24 h of incubation at 27℃ in Condition 2, but developed colonies in the first 24 h of incubation at 35℃ in Condition 1. Therefore, the author believes that Condition 1 is the preferred technique for rapid isolation of A. fumigatus.\n5) The MIC value for MCFG using the tentative endpoints was 0.0039-1 μg/ml against clinical isolates of Candida spp. in both methods, and the MIC against clinical isolates of Aspergillus spp. was 0.0078-0.0313 μg/ml by visual judgment using the CLSI method. A value of >4 μg/ml was judged using IC_80 as the endpoint and 0.0078-0.0313 μg/ml using IC_50 as the endpoint by spectrophotometry with the CLSI method, and a value of ≤0.0039-0.0156 μg/ml was judged with the JSMM method.\nIn the CLSI method, the absorbance of each well at the MIC in both quality control strains of Candida spp. decreased sharply, and the absorbance of the wells at or above the MIC were 0-1% of each growth control. On the other hand, in the A. fumigatus reference strain, the absorbance of the well at the MIC declined when judged visually, but the absorbance of the wells above the MIC was 28-48% of the growth control.\nWhen the MIC of C. krusei as quality control strain and A. fumigatus as reference strain were measured with the CLSI method, the microscopic morphology of the organisms in each well was examined simultaneously. The morphological changes in the organisms, the swelling of the cells in C. krusei and the rupture on the top of the hyphae in A. fumigatus were observed at half the concentration of each MIC. The transformed organisms in C. krusei were not observed in the wells at or above the MIC, but were observed in A. fumigatus up to the maximum concentration of MCFG.\nFor the observation of the rate of viable cells treated with each concentration of MCFG, neutral red staining was performed in each of the wells containing Candida spp. as the quality control, and A. fumigatus as the reference strain after measurements of MIC were carried out using the CLSI method. The rate of viable cells of Candida spp. as the quality control decreased to 2-6% of the growth control at concentrations above each MIC, and in A. fumigatus as the reference strain, the rate of viable cells declined at the MIC, but viable cells were observed at a rate of 12-19% of the growth control at concentrations above the MIC.\n6) To observe the effect of the inoculum size against MIC values, the inoculum size of the quality control and reference strains were investigated within a range of 10^2-10^6 cells /ml. MIC values for Candida spp. as the quality control were similar for all inoculum sizes. However, for A. fumigatus as the reference strain, the MIC at an inoculum size of 10^5-10^6 cells /ml was over 9 times higher than that for an inoculum size of 10^2-10^4 cells /ml.\n\nFrom these results, the author concludes that:\n1) The number of fungal colonies isolated from respiratory specimens was very low according to the mycological examination reports of the Kitasato University Hospital. Therefore, the author proposes that the treatment of clinical specimens, especially respiratory samples, is to dissolve the sputum with proteases, centrifuge the dissolved sputum and then use the pelleted sample for direct smear preparation and isolation. The author also proposes that large amounts of the inoculum at 30 μl should be added to the isolation medium, 35℃ incubation temperature for the first 24 h should be used for rapid isolation of C. albicans and A. fumigatus from clinical specimens. And the author also emphasizes that the incubation period for isolating fungi from clinical specimens requires 7 days for internal specimens and 21 days for dermatological specimens.\n\n2) After studying the susceptibility testing method for MCFG, the author concludes that the MIC measurement for MCFG against Candida spp. is acceptable by both CLSI and JSMM methods. However, when measuring MCFG against Aspergillus spp., the CLSI method has some problems. The inoculum size in the well of the microtiter plate should not exceed 10^4 cells/ml, and new endpoints should be used. This includes the visual endpoint provided in this study for visual judgment and the IC_50 endpoint by spectrophotometry. For MIC measurement of MCFG against Aspergillus spp. in the CLSI method, the author recommends the use of these new endpoints.\n","subitem_description_type":"Other"}]},"item_10006_description_7":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"真菌症の微生物学的検査（直接鏡検・分離培養）に関する検査法の標準化は遅れている。一般に真菌検査は、細菌検査に準じた方法で実施されているが、真菌は細菌より発育が遅いなどの特徴があり、必ずしも臨床材料からの真菌検出は十分でなく、真菌に適した検査法の確立が望まれている。そこで、臨床材料からの真菌の直接鏡検および分離培養検査の実態を把握するため、北里大学病院の臨床検査室で取り扱った真菌検査成績について、1) 直接鏡検の検出率、2) 呼吸器系疾患材料から分離された真菌（カンジダ属を除く）の菌種別分離頻度および集落数、3) 内科系疾患材料からのアスペルギルスおよびクリプトコックス分離例ならびに皮膚科材料からの皮膚糸状菌分離例における集落形成までの培養日数などを回顧的に調査した。さらに、カンジダ、アスペルギルスおよびクリプトコックスの保存菌株、各3株を用いて検体の前処理としての遠心分離の有用性、至適接種量および至適培養温度について検討した。\n　次に、抗真菌薬における最小発育阻止濃度〔minimum inhibitory concentration（MIC）〕の測定法は、米国のClinical and Laboratory Standards Institute（CLSI）が世界に先駆けて酵母用および糸状菌用の各検査法を発表したが、その後も改良が続く状況にあり、我が国では、日本医真菌学会（JSMM）がCLSI法を改良した検査法を提案している。2002年に新規の作用機作である真菌細胞壁中の（1→3）－β－D－グルカンの合成を阻害するキャンディン系抗真菌薬のミカファンギン（MCFG）が我が国で開発され上市されたが、キャンディン系薬のMIC測定法は制定されていなかった。\n　そこで、著者はCLSI法およびJSMM法の準拠を基本として、ヒト臨床材料由来のカンジダ48株、アスペルギルス13株、精度管理用菌株2株（Candida krusei ATCC6258、C. parapsilosis ATCC22019）および参照菌株（Aspergillus fumigatus ATCC26430）の計64株を供試して、MCFGのMIC測定法のうちMIC値への影響が大きい終末点の判定基準および適正接種菌量について検討した。\n　その概要は以下のとおりである。\n　1) 内臓アスペルギルス症と診断された27例中10例（37%）および内臓クリプトコックス症と診断された10例の12検体中10検体（83.3%）から、直接鏡検でそれぞれの真菌が確認された。\n　2) 呼吸器系疾患材料から分離されたカンジダを除く真菌69株の菌種別内訳は、Aspergillus fumigatus 43株（62.4%）、Asprgillus spp. 13株（18.8%）、Cryptococcus neoforｍans 10株（14.5%）およびCryptococcus spp. 3株（4.3%）であった。アスペルギルスが分離された102検体の分離集落数は、1～3集落のものが74検体（72.5%）と最も多く、数百集落以上は8検体（7.9%）に過ぎなかった。クリプトコックスが分離された14検体では、1～3集落が7検体（50.0%）、数百集落以上は2検体（14.3%）であった。\n　3) アスペルギルスが分離された65検体の初発集落形成までの培養日数は、1日が3検体（4.6%）、残り62検体（95.4%）すべてのアスペルギルス検出には6日必要であった。クリプトコックスが分離された3検体では、4日が1検体、5日が2検体であった。皮膚科材料から皮膚糸状菌が分離された46検体では、7日目までに集落を認めたのは29検体（63.1%）で、残り17検体すべての皮膚糸状菌検出には14日必要であった。\n　4) 保存菌株のカンジダ、クリプトコックスおよびアスペルギルスのそれぞれについて10～10^4cells/ml相当の菌液を作製し、遠心分離の有無による培養集落数を比較した。その結果、10cells/mlの菌液を遠心せずに一白金耳量（約10μl）塗抹した場合には集落が認められなかったが、2,000G、15分遠心後の沈渣を同様に塗抹したところ、1～3集落が認められた。また、10^2cells/ml相当の各菌液を用いて、1日目と2日目の培養温度をそれぞれ35℃、27℃とした場合、および2日間27℃で培養した場合の集落直径を比較した。その結果、前者の培養温度の方が、カンジダは1.2～1.4倍およびアスペルギルスは3.3～3.9倍大きくなったが、クリプトコックスは0.7～0.8倍と小さくなった。アスペルギルスは27℃、24時間培養では集落が認められなかったが、35℃、24時間培養で集落が認められたことから、35℃の培養条件はアスペルギルスの早期検出に有用であると考えられた。\n　5) MCFGのMIC測定時の終末点を仮設定後、MIC測定を行った結果、カンジダに対するMICはCLSI法およびJSMM法ともに目視および吸光度測定の両判定法で≦0.0039～1μg/mlとほぼ一致した。一方、アスペルギルスに対するMICはCLSI法の目視判定および吸光度を測定しIC_50による判定で、ともに0.0078～0.0313μg/mlを示したが、IC_80で判定した場合には>4μg/mlと大幅に異なる結果が得られた。JSMM法は両判定法ともに≦0.0039～0.0156μg/mlであった。その際のCLSI法での精度管理および参照菌株の各ウエルの吸光度値は、カンジダ2株ではMICより高濃度域で発育コントロールの0～1%の吸光度に激減したが、アスペルギルスは目視判定のMICより高濃度域で発育コントロールの28～48%の吸光度を示していた。そのためMIC測定後の各ウエル内の培養液中の菌体を光学顕微鏡で鏡検すると、目視判定の1/2MICでカンジダは菌体の膨化、アスペルギルスは菌糸先端の破裂などの形態変化（変形菌体）がそれぞれ認められたが、カンジダはMICより高濃度域で菌体を認めないのに対し、アスペルギルスは最高濃度まで変形菌体が認められた。\n　次いで、MIC測定後の各ウエルにニュートラルレッド液を添加してウエル内の生菌を染色し、その呈色液の吸光度を測定して各濃度別残存生菌量を測定した。その結果、カンジダはMICより高濃度域で発育コントロールの2～6%の生菌量であったが、アスペルギルスでは目視判定のMICより高濃度域で12～19%相当の生菌が残存することを確認した。\n　6) 10^2～10^6cells/mlの範囲で適正接種菌量を検討した結果、カンジダはCLSI法およびJSMM法ともに、いずれの接種菌量でも同じMICを示した。アスペルギルスはJSMM法ではいずれの接種菌量もほぼ同じ値を示したが、CLSI法では10^5cells/ml以上では、それ以下の接種菌量より9管以上高いMICを示した。\n　以上のように、臨床検査成績の精査によって、呼吸器系疾患材料に含まれる真菌は少量であることが明らかになった。喀痰を検体とする場合はプロテアーゼなどの溶解剤を用いて液化後、遠心分離した沈渣を塗抹標本および分離培養の試料とすること、分離培養の接種量は一滴（約30μl）の大量とすること、アスペルギルスおよびカンジダを対象とする場合は初日の培養温度を35℃とすること、培養期間は内科系疾患材料では7日、皮膚科材料は3～4週間は必須であることを明らかにした。\n　また、MCFGのMIC測定法は、カンジダはCLSI法およびJSMM法のいずれでも問題がないが、アスペルギルスは多用されるCLSI法で問題が多く、接種菌量は10^4cells/mlを越えないこと、目視判定では「ウエル内の菌塊から菌糸の発育を認めない」点、吸光度測定ではIC_50を、あらたな終末点とする必要性を明らかにした。\n","subitem_description_type":"Abstract"}]},"item_10006_dissertation_number_12":{"attribute_name":"学位授与番号","attribute_value_mlt":[{"subitem_dissertationnumber":"乙第22号"}]},"item_10006_version_type_18":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_ab4af688f83e57aa","subitem_version_type":"AM"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"阿部, 美知子"}],"nameIdentifiers":[{"nameIdentifier":"17721","nameIdentifierScheme":"WEKO"}]},{"creatorNames":[{"creatorName":"Abe, Michiko","creatorNameLang":"en"}],"nameIdentifiers":[{"nameIdentifier":"17722","nameIdentifierScheme":"WEKO"}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2014-04-16"}],"displaytype":"detail","filename":"diss_de_otsu0022.pdf","filesize":[{"value":"1.9 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_de_otsu0022","url":"https://az.repo.nii.ac.jp/record/3834/files/diss_de_otsu0022.pdf"},"version_id":"9d741b09-db84-4849-84ad-212851dfc5bb"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2014-08-19"}],"displaytype":"detail","filename":"diss_de_otsu0022_jab&rev.pdf","filesize":[{"value":"382.0 kB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"diss_de_otsu0022_jab&rev","url":"https://az.repo.nii.ac.jp/record/3834/files/diss_de_otsu0022_jab&rev.pdf"},"version_id":"e75ed3bf-041a-4192-96ac-ed9840114529"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"真菌症の臨床微生物学的検査法，とくに検体の前処理，分離培養およびミカファンギン(MCFG)の感受性測定法の確立に関する検討","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"真菌症の臨床微生物学的検査法，とくに検体の前処理，分離培養およびミカファンギン(MCFG)の感受性測定法の確立に関する検討"},{"subitem_title":"Study on the establishment of mycological examination methods in clinical laboratories : specimen pretreatment and isolation and a susceptibility testing for micafungin","subitem_title_language":"en"}]},"item_type_id":"10006","owner":"4","path":["376"],"pubdate":{"attribute_name":"公開日","attribute_value":"2014-01-10"},"publish_date":"2014-01-10","publish_status":"0","recid":"3834","relation_version_is_last":true,"title":["真菌症の臨床微生物学的検査法，とくに検体の前処理，分離培養およびミカファンギン(MCFG)の感受性測定法の確立に関する検討"],"weko_creator_id":"4","weko_shared_id":4},"updated":"2023-06-19T08:13:30.787745+00:00"}