| Invasive fungal disease (IFD) are a growing problem in severely immunocompromised patients suffered from hematologic malignancy, solid organ transplant, hematopoietic stem cell transplant and other critical illness receiving corticosteroids, cyclosporine, and other immunosuppressive treatment. Aspergillus.fumigatus and Candida. albicans is the most common species isolated from patients with IFD. Sporothrix globosa is pathogenic agent of sporotrichosis which is a common subcutaneous infection in China. As fungal species vary in their susceptibility to antimycotic agents, accurate species diagnosis is essential in IA to guide antifungal therapy and improve the prognosis. However, conventional approaches, including microscope, culture and histology, lack efficiency and, sometime inaccuracy in diagnosis. In order to overcome the drawbacks of traditional fungal examination, Laser capture microdissection (LCM) technology has been developed to facilitate diagnosis of IFD. To validate the assays and understand the potential diagnostic utility of LCM in diagnosis of IFD, here, we microdissected and collected hyphal strands or spores directly from tissue of three murine model of IFD, challenged by A.fumigatus, C.albicans and S. globosa respectively, using LCM, and subsequently processed for DNA extraction and PCR/nested PCR-sequencing. The current research was divided into three parts as follows:Chapter one:the Application of Laser Microdissection in Molecular Detection and Identification of Aspergillus fumigatus from Murine Model of Acute Invasive Pulmonary AspergillosisBalb/c mice were infected intranasally with1×108CFU/ml of A. fumigatus. After mice were sacrificed, lungs were harvested and divided into three parts. The first part of the specimens was fixed in90%ethanol, subsequently stained with HE and PAS. The second part was cultured on SDA at30℃to morphological identification and extraction of DNA of the A. fumigatus strain for nested PCR (n-PCR) amplification as positive control. The third part was frozen and sectioned. We microdissected and collected Blankophor-stained20-30μm(25.7±3.0μm) length single or three hyphal strands from tissue crysections of murine model of IPA by LCM, subsequently processed for DNA extraction, and amplify the18S rRNA region of A.fumigatus by n-PCR; subsequently, sequencing, and species molecular identification. In single hypha group,18of20microdissected hyphal strands were identified as A. fumigatus.In three hyphae groups, 20of20microdissected hyphal strands were identified as A. fumigatus. The sensitivity of LCM-n-PCR-sequencing in these two group was90%and100%respectively, there was no significant difference between them (χ2=0.489, P)0.05).And totally sensitivity and specificity was95%and95%.The result of our research was more sensitive than the previous report.Chapter two:The Application of Laser Microdissection in Molecular Detection and Identification of Candida.albicans from Murine Model of Acute Invasive CandidiasisBalb/c mice were infected by inoculation of0.5ml of1×106CFU/ml of C. albicans in the tail vein. After mice were sacrificed, kidney were harvested and divided into three parts for HE/PAS stain, cultured on SDA as positive control, and crysectioned, respectively. We microdissected and collected Blankophor-stained single or five spores from tissue crysections of murine model of IC by LCM, subsequently processed for DNA extraction,and amplify the5.8SrRNA region of C. albicans by n-PCR; subsequently, sequencing, and species molecular identification. In single spore group,25of30microdissected spores were identified as C.albicans.In five spores group,27of30microdissected spores were identified as C. albicans.The sensitivity of LCM-PCR-sequencing in these two group was83.3%and90%respectively, there was no significant different between them (χ2=0.71, P)0.05).And totally sensitivity and specificity was87.7%and95%. The result point to a high sensitivity and specificity of the method in animal mode.Chapter three:the Application of Laser Microdissection in Molecular Detection and Identification of Sporothrix globosa from Murine Model of Acute Invasive Sporotrichosis and clinical specimensBalb/c mice were intratesticularly infected by inoculation of25μl of1×106CFU/ml of S.globosa in the tail vein. After mice were sacrificed, testicle were harvested and divided into two parts for HE/PAS stain, cultured on SDA as positive control, respectively. We microdissected and collected PAS-stained single or five spores from tissue sections by LCM, subsequently processed for DNA extraction, and amplify Chitin synthase gene I by PCR, and the ITS2region by n-PCR, respectively; subsequently, sequencing, and species molecular identification.In PCR group, single spores subgroup,66.67%(20/30)LCM samples was identified as S.globosa,in five spores subgroup,77.67%(23/30)LCM samples were identified as S.globosa,there was no significant difference between them(χ2=0.567, P)0.05). In n-PCR group, the sensitivity of single spore and five spores were70.00%(21/30)and83.33%(25/30)respectively, there was no significant different between them(x2=0.360, P>0.05).The specificity was100%. Meanwhile,we retrospectively detected the8archive skin specimens of patients with sporotrichosis by LCM.We microdissected1spore on PAS stained specimens and then amplification by PCR/n-PCR.Among the8archive specimens,3were positive by both PCR and n-PCR,1specimen was just positive by n-PCR. The result suggest that pretreatment of sample may play a key role in the diagnosis and detection of IFD by assay of LCM in conjunction with PCR/n-PCR.Otherwise, the amount of hyphae/spores and the different amplification method of DNA could influence the result in some degree.In conclusion, we assessed the value of validation of the LCM in conjunction with PCR/n-PCR sequencing method in molecular detection and identification of pathogenic fungi in murine model of IFD. The result pointed to a high sensitivity and specificity of the test in animal model, promising use in the clinically attempts to improve diagnosis of IFD, potentially decreasing the chances of samples contamination or colonization. Moreover, the result of the method concurrently identified the pathogenic fungus to species level which is imperative in association with the selection of sensitive antifungal drug. So, the use of LCM based method could be a powerful tool for development of new assays in diagnosis of IFD. |
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