侵犯性真菌病於免疫功能低下、免疫功能抑制以及重症病人有很高的發生率、致死率以及死亡率。受感染病人的低存活率，部分歸咎於早期診斷和治療侵犯性真菌病的困難性；感染侵犯維生器官的各種真菌不但生長緩慢而且具有不同的抗藥性。常見致命真菌菌種有較高的分離率以及致病性，而抗真菌藥物是有效治療這些真菌感染的方法；然而不同的真菌菌種對某些抗真菌藥物具有不同的感受性，而無法以這些藥物治療所有的真菌感染。目前這類疾病的傳統診斷方法（例如臨床症狀、影像診斷以及生化免疫試驗），其特異性仍不足以區別診斷不同的致病真菌至屬或種的層次。顯微鏡下的形態學鑑定可區別診斷某些致病真菌菌屬；然而這些方法需培養較久的時間、鑑定不易、很難準確地診斷致病真菌菌種。
　　本論文的研究動機為：解決傳統鑑定/診斷真菌的困境，避免因延遲或不準確的診斷而造成感染致命真菌病人的死亡；本論文的研究目的為：提出質譜與拉曼散射光譜搭配互補的方法，以適用於培養早期之各個分化階段進行致病真菌菌種診斷及抗真菌藥物敏感菌種篩檢；並提供較多樣的分子資訊，以區別診斷不同的致病真菌至種的層次。本論文所使用的真菌為：先以來自台灣或荷蘭菌種中心的參考菌株建立真菌菌種的質譜與拉曼光譜資料庫，再以來自台灣（台南成大醫院、花蓮慈濟醫院）、英國（Leeds 醫院）及法國（Angers 醫院）的臨床檢體，以及其它參考菌株進行測試。本論文的研究過程：首先發展適合真菌培養初期之不同階段檢測的質譜或拉曼光譜感測技術；接著統計分析這些波峰譜線的再現性、特異性與重疊性、偏離容忍度等，以建立不同菌種之特徵峰資料庫並建立峰值比對及菌種診斷準則；然後再以臨床及參考菌株評估其診斷能力。樣本的光譜經品質管制，訊號不佳的樣本需重做實驗或剔除，以避免因訊號不佳所造成的診斷能力下降。本論文之研究成果共分為五項：
　　（一）發展質譜感測技術供早期鑑定常見致命麴菌菌種：三氟醋酸蛋白質萃取法適用於麴菌菌種的質譜檢測；其可於孢子形成的第一階段（培養 1–2 天）與第二階段（培養 2–4 天）進行鑑定。孢子形成的第二階段含有較多不同分化細胞的蛋白質分子特徵峰可供比對鑑定。經 16 株來自菌種中心的參考菌株及 10 株來自不同醫院臨床檢體的真實樣本之評估，質譜的重疊峰與特徵峰資料庫搭配合適的鑑定標準（大於 50% 的符合率）可正確地鑑定常見致命麴菌至「種」的分類層次。
　　（二）發展非破壞性之單細胞之顯微拉曼光譜感測技術以供孢子開始形成時（培養 2 天）鑑定診斷三種常見致命麴菌菌種（薰煙麴菌、黑麴菌以及黃麴菌）：單一孢子經簡單塗抹分離而得，並固定在鍍金的載玻片上。成熟孢子可由亮視野顯微鏡下的深色灰階影像或明顯的共振或一般拉曼光譜鑑別；這些訊號可能來自孢子的天然色素、蛋白質及核酸等分子。經 32 株參考菌株及 19 株臨床檢體等真實樣本評估，光譜的重疊峰與特徵峰資料庫搭配合適的鑑定標準（大於 50% 的符合率）可精確地鑑定念珠菌到「種」的分類層次；其靈敏度為 100%，而特異性為 88%–100%。
　　（三）發展 MALDI-TOF 質譜感測技術以供早期（培養 1–2 天）鑑定致命念珠菌菌種：加熱去活化結合甲酸蛋白質萃取法較適用於念珠菌的 MALDI-TOF 質譜檢測。取自菌落的足夠酵母菌細胞（> 106 cells/mL）可以被測出明顯的質譜訊號；而取自陽性血瓶的足夠真菌細胞之質譜訊號則易受血液成分所干擾與抑制。固態培養的念珠菌形態一致（酵母菌型），其質譜的再現性與特異性較高。而陽性血瓶培養的念珠菌型態則不一致（含酵母菌、假菌絲及真菌絲），其質譜的特異性較低。經 19 個參考菌株的階層群集分析，以及 30 株菌種中心樣本與 21 株來自不同醫院的臨床樣本進行評估，質譜資料庫搭配適合的鑑定標準（比對到 8 個特徵峰以上）可鑑定診斷致病念珠菌到「種」的層次。
　　（四）發展拉曼光譜感測技術以供增長初期（培養 1–2 天）進行鑑定致命白色念珠菌種：單一酵母菌型態活菌可經由固態培養或是添加 0.125 μg/mL flucytosine 之液態培養（於 63 小時內無細胞毒殺效應）而取得，並固定在鍍金玻片或矽晶片上以避開紅外光雷射對玻璃所產生的螢光。以拉曼光譜圖形結合多變數分析法（主成分分析及階層群集分析），可將具有相似波譜之 6 種酵母菌（共 7 株）分為三群。經 13 個樣本評估，以光譜圖形結合特徵峰比對（比對到 1 個特徵峰以上）之鑑定法，則可鑑定白色念珠菌到「種」的層次。
　　（五）發展表面增顯（共振）拉曼光譜感測技術，以供生長初期（培養 1–2 天）篩檢核黃素陽性（fluconazole 敏感性）念珠菌菌種：首先於固態培養早期的可見菌落取得足夠的酵母菌細胞（> 108 cells/mL），並和高濃度的奈米銀粒子混合以快速檢測並增強細胞表面分子的拉曼光譜訊號。接著以多變數分析法（主成分分析及階層群集分析）區別不同菌種的相似光譜；然而，部分菌種可能因為不同的細胞表面分泌物所造成的訊號干擾，而無法清楚地鑑別到「種」的層次。此外，念珠菌被培養於相對厭氧環境及缺鐵培養液中以進行增長與醱酵，並於液態培養的指數增長期取得足夠的菌絲及酵母菌細胞。經去除培養液後，以高濃度奈米銀粒子檢測與增強微量殘留在菌體表面分子的拉曼光譜訊號。然後再和核黃素的特徵峰資料庫比對，並以合適的鑑定標準（大於 50% 的符合率）篩檢出具有明顯核黃素訊號的真菌樣本。經 14 株臨床檢體評估，此二分法可正確地鑑別核黃素陽性（fluconazole 敏感性）念珠菌與核黃素陰性（fluconazole 抗藥性）。
　　這個方法可提供豐富的分子資訊，以鑑定致病真菌至種的層次；其亦可於真菌緩慢生長的初期檢測分子表現，以縮短傳統形態學鑑定與藥物感受性試驗所需之冗長培養時間。

　　Invasive mycoses have high incidence, fatality, and mortality in immunocompromised, immunosuppressed, and critically ill patients. The low survival rate is partially attributed to the difficulties of early diagnosis and treatment of these diseases; various lethal fungal species invading vital organs not only grow slowly but also have different drug resistance. Common lethal fungi have higher isolation rate and virulence than other fungal species and some of them can be treated by antifungals; however, there are different susceptibilities of different fungal species against certain antifungals. It is difficult to effectively treat all fungal species using these drugs. Current diagnostic methods (such as clinical manifestations, radiological images, and biochemical/immunological tests) are not specific enough to differentially diagnose these pathogenic fungi to the genus or species level. Morphological features of colonies and microscopic images can differentially identify some pathogenic fungi to the genus level; however, they are very time-consuming, and difficult to accurately diagnose these fungi to the species level.
　　The motivation of this dissertation is to resolve the dilemma of traditional diagnostic methods and then prevent the death of fungus-infected patients owing to the delayed and inaccurate diagnosis and treatment. The purpose of this dissertation is to propose a complementary method based on MALDI-TOF mass spectrometry and Raman spectroscopy for early identification of different lethal fungal species (Aspergllus and Candida) and screening antifungal-sensitive species at different differentiated phases of the early growth stage; besides, the purpose is to provide plentiful molecular information to accurately identify common pathogenic fungi to the species level. The reference strains for establishing spectral databases were purchased from the collection centers of Taiwan (BCRC) and Netherlands (CBS); clinical specimens were obtained from hospitals in Taiwan (Cheng Kung University Hospital and Tzu Chi General Hospital), UK (Leeds Hospital), and France (Angers Hospital). The research process is briefly described as follows. First, appropriate bio-sensing approaches based on MALDI-TOF spectrometry and Raman spectroscopy were developed to fit the detection of different phenotypes of different fungi at different early growth stages on solid agar plates or liquid media. Then, the strain-to-strain reproducibility, species-to-species specificity/overlap, and the tolerance of deviation were statistically analyzed to characterize and establish spectral databases of different fungal species; besides, the criteria for identifying identical peaks and diagnosing fungal species were established. Furthermore, real samples from collection centers and clinical specimens from hospitals were used to evaluate the diagnostic performance of the criteria. The spectral quality of test samples was controlled. The samples with very weak spectral signals were improved/acquired by appropriate experimental parameters again or deleted in order to prevent the decrease of diagnostic performance owing to the poor signal quality. Major results of this dissertation are divided into five parts:
　　(1) Development of mass spectral sensing technologies for early identification of common lethal Aspergillus species: Trifluoroacetic acid is more suitable for extracting fungal proteins of Aspergillus for MALDI-TOF mass detection. These proposed approaches based on MALDI-TOF mass spectra can be used to identify different Aspergillus species at the early growth stage I (culturing for 1–2 days) and stage II (culturing for 2–4 days) of fungal sporulation. There are more species-specific peaks originating from various differentiated cells at the growth stage II of sporulation for comparison and identification. The criteria (> 50% matching rate of overlapping/specific peaks in the database) can be used to identify common lethal Aspergillus to the “species” level from the established spectral database; this was demonstrated by testing 16 reference strains from the collection center and 10 clinical specimens from hospitals.
　　(2) Development of nondestructive approaches based on Raman microspectroscopy to detect a single spore at the onset of sporulation (culturing for 2 days) and species diagnosis of A. fumigatus, A. flavus, and A. niger: A single conidiospore was simply prepared by scratching the surface of colonies, diluting them in sterile de-ionized water, smearing the suspension on a gold-coated glass slide, and air drying to immobilize spores. The mature single spores of A. fumigatus, A. flavus, and A. niger can be identified by dark appearances under bright-field microscopes or prominent resonance/normal spectra; the characteristic spectral peaks of these Aspergillus species were tabled to establish the reference database for identification of real samples; these signals may be originating from natural pigments, proteins and nucleic acids. The criteria (> 50% matching rate of overlapping/specific peaks in the database) can identify pathogenic Aspergillus to the “species” level with high sensitivity (100%) and specificity (88–100%) in the evaluation of 32 reference strains from the collection center and 19 clinical specimens from the hospitals.
　　(3) Development of MALDI-TOF mass bio-sensing technologies for early identification of lethal Candida species at early growth stages (culturing for 1–2 days): Wet-heating inactivation combined with formic-acid extraction of proteins is more feasible for MALDI-TOF mass detection of Candida species. Enough yeast cells (> 106 cells/mL) acquired from the early stage of visible colonies cultured on agar plates can be detected by MALDI-TOF mass spectrometry; in contrast, MALDI-TOF mass signals of enough fungal cells acquired from the blood bottles are inhibited by the components of blood. There is a uniform phenotype (yeast-like cells) of Candida species culture on agar plates; MALDI-TOF mass signals of them are strain-to-strain reproducible and species-to-species specific. In contrast, there are various phenotypes (yeast, pseudohyphae, and true hyphae) of Candida species culture in the blood bottles. MALDI-TOF mass signals of them have low reproducibility and low specificity. Databases of species-specific mass peak were established to identify different Candida species. The appropriate criteria (> 8 matched peaks of specific peaks in the database) can identify Candida to the “species” level with high sensitivity (100%) and specificity (100%) by the evaluation of 30 reference strains from different collection centers and 21 clinical specimens from different hospitals.
　　(4) Development of Raman bio-sensing technologies for early identification of lethal Candida at early growth stages (culturing for 1–2 days): Single yeast cells can be acquired from visible colonies on agar plates and flucytosine-treated Candida. Flucytosine with the concentration of 0.125 μg/mL can prevent the formation of hypha-like cells of Candida species cultured in liquid media without significant cytotoxic effect until the treatment for 63 hours. Single yeast cells were immobilized on silver-coated electrodes on glass slides to avoid strong fluorescence of glass induced by an NIR laser. Six different yeast-like fungal species (7 strains) can be discriminated into three groups by their spectral patterns with multivariate analysis (principal component analysis and hierarchical cluster analysis). By the evaluation of 13 test fungal samples, C. albicans can be further identified by comparing both the spectral pattern and the species-specific peak (> 1 matched peak of specific peaks in the database).
　　(5) Development of surface-enhanced (resonance) Raman bio-sensing technologies for screening riboflavin-positive (fluconazole-sensitive) species of Candida at the early growth stages (culturing for 1–2 days): First, enough yeast cells (> 108 cells/mL) acquired from visible colonies on agar plates were mixed with high-concentration silver nanoparticles for rapid detection and enhance Raman signals of molecules on the cell surface. Multivariate data analysis (principal component analysis and hierarchical cluster analysis) was used to discriminate similar spectral patterns of different species; however, partial strains which cannot be clearly discriminated to the “species” level may be due to the interference of various secreted molecules on the surface of yeast-like cells. In addition, Candida was cultured in liquid media (lack of ion) under a relatively anaerobic condition for fermentation and growth. Enough hypha-like and yeast-like cells were acquired from the log phase in liquid media. After washing out growth media, high-concentration silver nanoparticles were used for rapidly probing trace residual molecules on the cell surface and enhancing their Raman signals. Then, these spectral peaks were compared with the database of specific peaks of riboflavin. The fungal sample which has strong SERS signals of riboflavin was identified as riboflavin-positive by the appropriate criterion (> 50% matching rate of SERS peaks of riboflavin). Riboflavin-positive species (fluconazole-sensitive) and riboflavin-negative species (fluconazole-resistant) species of Candida can be correctly classified by the evaluation of 14 clinical specimens cultured in liquid media.
　　In conclusion, this new complementary method can provide rich molecular information to identify/diagnose pathogenic fungi to the species level. In addition, this can detect the molecular expression at early stages of the slow fungal growth to effectively shorten the long-term fungal culture of traditional morphological identification and susceptibility testing.

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