Rapid Detection Of Penicillium Marneffei In Serum From AIDS Patients By Enzyme-linked Immunosorbent Assay

Background and Objective: Penicillium marneffei (P.m), which is the only known temperature dependent dimorphic species among the genus Penicillium, is a common opportunistic pathogen seen in AIDS patients. The diagnosis of Penicilliosis marneffei (PSM) should be confirmed either by mycological inoculation or histological examination. But it costs several days to get results by fungal culture, histological examination, however, sometimes get false negative, and several intracellular pathogens bring challenge to differentiation. The molecular techiniques like PCR and DNA microarray have showed some advantages on diagnosis, but still need more clinical validation. We present here new way to detect P.m antigen by using prepared P.m specific antibody.Methods: (1) As co-workers of the human genome research center of China in Shanghai, we have analysed the functional genes of P.m by methods of molecular biology and bioformatics. Several cDNA librarys of P.m were both constructed and analysed and two new specific genes of P.m PYA₀24₆0 and PYA₀12₉2 which are likely to code albumen on cell wall were found. We immunised Rabit of New Zealand with producted proteins which came from Escherichia coli through gene expression. So two specific polyclonal antibody came into being. PYA₀12₉2 antibody which is more specific was selected for our research. (2) twenty-three archived AIDS complicated with PSM cases were selected, and 4μm thick series sections were continuously cut from each paraffin-embedded PSM tissues block, these sections were stained by hematoxylin and eosin(HE), Periodic Acid Schiff (PAS) and immunhistochemical staining using prepared P.m antibody,respectively. 3 cases of actinomycetes and 20 cases of other mycotic infections were collected as control, including 14 cases of aspergillus, 2 cases of Candida albicans, 2 cases of Cryptococcus neoformans, 2 cases of mucor infections. (3)P.m antibody was labeled with HRP by NaIO₄ method. Double immunodiffusion was performed with HRP- P.m antibody which had been purified and serum from PSM patients to validate the activity of HRP- P.m antibody. (4) The experimental protocol was set up through coating nitrocellulose membrane with P.m antibody, then applicated to detect serum from AIDS patients complicated by Penicillium marneffei infection with Dot-ELISA .(5) P.m antigen in sera from 103 cases of AIDS patients were detected by ELISA double sandwich method and Dot-ELISA.Results: (1) Microscopically, all 23 cases of PSM had AIDS settings, the normal structures of their lymph nodes were almost completely erased by striking proliferating macrophages. PAS and D-PAS stainings revealed the characters of P.m, including the specific 'sausarge cell ', and cross-septum of the cell, consistening with the primary diagnosis. (2) P.m from 22 cases of PSM were positive by immunohistochemistry staining, the positive reaction on the wall of fungi were stronger than that in the cytoplasm. However, no positive reaction was seen in all controls. (3) Double immunodiffusion shows that HRP- P.m antibody is full of activity of either HRP or antibody. (4) There was no significant of differences of the results get by ELISA and Dot-ELISA. The sensitivity and coincidence rate of the ELISA and Dot-ELISA are 75% and 97.09%, 87.5% and 98.06%, respectively. ELISA is better than Dot-ELISA.Conclusions:1 The detection of antigen of Penicillium marneffei in serum by ELISA is with sensitivity, specificity and effectivity, which can be applicable to general clinical laboratories for scaning and definitive diagnosis of the infection.2 The ELISA double sandwich method of qualitative detection has been set up to detect P.m in serum, which is prepared for quantitative detection and manufacture of the enzywell P.m kit.