| Aspergillus fumigatus is a ubiquitous filamentous fungus in the environment. On average, humans inhale hundreds of Aspergillus conidia daily. These conidia are eliminated efficiently by innate immune mechanisms in immunocompetent hosts, but could germinate and colonize in immunocompromised host. As a result of the increasing use of transplantation for end-organ disease, the use of immunosuppressive and myeloablative therapies for autoimmune and neoplastic diseases, and the human immunodeficiency virus (AIDS) pandemic, the number of immunocompromised patients is steadily expanding. In these patients, the conidia of A. fumigatus, with diameters (3to5μm) that allow them to traverse the terminal respiratory airways and reach the pulmonary alveoli, could escape the defense line and germinate in the lung, causing pulmonary aspergillosis. In most cases, the fungal pathogen will also migrate to other organs, such as myocardium, kidney, liver, spleen, soft tissue, and bone, causing invasive aspergillosis (IA)--a severe and usually fatal systematic infection. The incidence of IA can reach as high as50%in acute leukemia if the patients have a risk of Aspergillus propagation and dissemination, such as during building/ construction work. The mortality rate is often around50%. The eary diagnosis is very impotant for IA patients。Since the signs and symptoms of IA are nonspecific, early clinical diagnosis of the infection is often difficult. The "gold standard" for diagnosis is to obtain a positive culture of A. fumigatus and to obtain histological evidence of mycelial growth in biopsy specimens. However, owing to its invasiveness, biopsy is often precluded from patients in critical conditions. A. fumigatus cultures from blood or respiratory specimens are often false-negative, especially during early stages of the disease. As an adjunctive measure to microbiological methods, antibody detection is usually performed for the diagnosis of IA, but these assays are usually insufficient owing to limits in their sensitivity and specificity. False-negative results are often come out because of the fulminant nature of the disease and the poor immunological status of the host, and false-positive results also exist in healthy individuals because of the prolong interaction with conidia. At present, most of the studies focused on the identification of antigenic markers and the DNA targets of IA.At present, the diagnostic value of galactomannan (GM), a major component of the Aspergillus cell wall, has been identified. Platelia Aspergillus (Bio-Rad, Marnes-La-Coquette, France) assay, an immunoassays that employ a rat immunoglobulin M (IgM) Monoclonal antibody (MAb) designated EB-A2for the detection of circulating Aspergillus GM have been developed, and been widely used in many countries, including USA. Although previous studies that assessed the performance characteristics of this assay reported high sensitivity and specificity, more recent studies have shown that many significant variations indeed come out by using this method. The causes of this variability are multifactorial and, in large part, cannot be explained because there is insufficient understanding of the kinetics of GM in vivo. Besides GM,(1,3)-(3-D glucan (BG) antigen is another important marker for the early serodiagnosis of deep-seated fungal infections. However, BG capturing assay cannot be used for identification at the species level, because BG is a major component of the fungal cell wall, including Aspergillus, Conidia, et al. So, the identification of other antigenic markers, may contribute to the development of new antigenic capture assays.In2005, a whole-genome shotgun approach was finished, and the complete genomic sequence of A. fumigatus strain Af293was obtained. These made designing specific primers based on sequence alignment and genemic data very conveniently, and many researchers pay their attention to develop biomolecular diagnosis of IA. Although there are still many problems of using biomolecular diagnosis in clinic, including difficulties in standardization, biomolecular diagnosis also has many advantages, such as the accurate classification of different types in the same strain, and the acquaintance of drug resistance. Therefore, the establishment of Aspergillus specific biomolecular diagnositic method can contribute to the implementation of IA early rapid diagnosis.In this study, we managed to build antigen-capture ELISAs and biomolecular diagnosis of I A.1. Expression of recombinant Afmplp using pichia expression system, preparation of murine anti-Afmplp MAbs, and development of Afmplp-capture ELISA for diagnosis of A. fumigatus infectionAs one of the galactomannoproteins in A. fumigates, Afmp1p is a homolog of Mplp, which is a highly antigenic cell wall mannoprotein in P. marneffei and was found to be a very useful target for the serodiagnosis of P. marneffei infections. It is speculated that Afmp1p has the same potential for use in aspergillosis serodiagnosis. Previously, we developed antigen and antibody tests using E.coli expressed GST-Afmplp and guinea pig anti-GST-Afmplp PAbs. Although no false-positive results were found in serum samples from negative control groups, sensitivity of the assays were found to be relatively low in which only8of15(53%) IA patients were Afmp1p antigen test positive. To achieve higher sensitivity and to lower the inter-and intra-laboratory variations introduced during the collection of guinea pig anti serum, MAb-based immunodiagnostic assays was proposed. A GST-Afmplp-capture ELISA using murine MAbs was developed with sensitivity of0.1ng/ml, however, it failed to capture natural Afmplp from infected sera. The failure to detect natural form of Afmplp by ELISA indicated that differences between GST-Afmplp and natural Afmp1p existed. To exclude the potential defects in prokaryotic expression systems, we selected the Pichia expression system to express Afmplp, and attempted to build an Afmplp-capture ELISA to detect A. fumigates infection.2. Expression of recombinant Afmp4p using pichia expression system, preparation of murine anti-Afmp4p MAbs, and development of Afmp4p-capture ELISA for diagnosis of A. fumigatus infectionLike Afmplp, Afmp4p is another homolog of Mplp, and may have potential for serodiagnosis of aspergillosis. In this study, besides Afmplp, we selected the Pichia expression system to express Afmp4p too, and tried to build an Afmp4p-capture ELISA to detect A. fumigates infection.3. Development of rDNA ITS real-time PCR for diagnosis of A. fumigatus infectionRibosomal DNA (rDNA) codes for ribosomal RNA. The rDNA gene cluster from5’ to the3’ are the18S rDNA, internal transcribed spacer1(internal transcribed space, ITS1),5.8S rDNA, ITS2,28S rDNA. Among these regions,18S rDNA and ITS, consists of conserved regions and variable regions, and are good markers for biomolecular diagnosis in clinic.In this study, we selected two pairs of PCR primer, one is pan-fungal rDNA ITS primer (ITSS:5’-GTGAATCATCGAATCTTTGAAC-3’, ITSR:5’-TCCTCCGCTT ATTGATATGC-3’), the other one is pan-fungal18S rDNA primer (18SS:5’-ATTGG AGGGCAAGTCTGGTG-3’,18SR:5’-CCGATCCCTAGTCGGCATAG-3’). Using the Aspergillus genomic DNA as templates, we obtained two types of different PCR products. After direct sequencing and gene sequence analysis, we seleced the better sequence for real-time PCR target, designed real-time PCR primer and probe, established a real-time PCR detecting method, but the sensitivity of this method is not high enough. In order to solve this problem, we subquently established a nested real-time PCR.Conclusion1. We expressed galactomannoprotein Afmplp, a potential bio-marker of A. fumigates infection. The purified rAfmplp showed as a band of proximate40kDa in western blotting, bigger than anticipation. This maybe caused by glycosylation, since Afmplp has potential O-glycosylation sites, and is expected to be a glycosylated protein. Twenty hybridoma cell lines each producing different MAbs against Afmplp were established, and were named from Afmpl-M1to Afmpl-M20. To select the optimal capturer-detector for a MAb-MAb ELISA to detect Afmp1p in A. fumigatus, the sensitivities of all possible pairs of MAbs to against rAfmp1p and native Afmp1p were determined, and Afmp1-M3and Afmp1-M6-HRP were selected as the capturing and detecting antibody for the Afmp1p capture ELISA. This ELISA would be positive even when the culture of A. fumigatus had been diluted into128-folds of its original concentration. And the limit for rAfmplp detection was approximately400pg/ml. The ELISA could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and had no cross-reactivity to other Aspergillus challenged animal models. Afmp1p-capture ELISA may be useful in clinical diagnosis of aspergillosis. 2. A potential bio-marker of A. fumigates infection-galactomannoprotein, Afmp4p was expressed. The purified rAfmp4p showed as a band of proximate19kDa in western blotting. Thirteen hybridoma cell lines each producing different MAbs against Afmp4p were established, and were named from Afmp4-M1to Afmp4-M13. Afmp4-M9and Afmp4-M2-HRP were selected as the capturing and detecting antibody for the Afmp4p capture ELISA. This ELISA would be positive even when the culture of A. fumigatus had been diluted into512-folds of its original concentration, and the limit for rAfmp4p detection was approximately800pg/ml. This ELISA could capture circulating or excreted antigens during the acute phase of invasive aspergillosis (IA) in the animal model, and no cross-reaction to the other Aspergillus infected rabbit sera and the sera prior infections had been observed.3. Among these two pan-fungi PCR primers, pan-fungal18S rDNA PCR products analysis founded that all of them have the same sequence in different fungi, and pan-fungal ITS PCR products had different sequence in different fungi. These results indicated that fungal ITS region is a better target for diagnosis in species level. Though analysis of different fungal ITS sequence, we designed real-time PCR primer, the forward primer is5’-TATGGGGCTTTGTCACCTC-3’,the reverse primer is5’-TCCTCCGCTTATTGATATG-3’, and the Taqman MB probe is5’-CCG GCGCCAGCCGACACCCAACTTTA-3’. Using this real-time assay, we can detect A. fumigates, A. flavus and A. terrus, but the sensitivity of this method is not high enough. Sebquently, a nested real-time PCR was been built, with a pan-fungal ITS PCR augmentation before real-time PCR. This method can sepecif test the Aspergillus DNA, and other fungi, including A. nidulans, A. niger, Penicinnei marneffei, and the Condidia have negative results using this method.In conclusion, we successfully established two antigen-capture ELISAs and a rDNA ITS detection to specifically detect A. fumigates infections in the present study. Further studies will focus on evaluating the efficacy of this Aspergillus antigen assay with clinical samples. |
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