Experimental Study Of Fungal Endophthalmitis

In recent years, along with the extensive use of the broad-spectrum antibiotics,the immunosuppressant and the corticosteroid on the clinic, the prevalence of theorgan transplantation and the indwelling catheters continuously, and the increasingpatients of the AIDS and diabetes mellitus, the fungal endophthalmitis has presentedan increasing trend in the proportion of infectious endophthalmitis.Fungalendophthalmitis is a kind of infectious disease inside the eyeball caused by thesuperficial ,the deep fungus or the conditioned pathogen and have a lower incidencerate than the bacterial endophthalmitis.It is a kind of severe eye disease leading toblind. Aspergillus fumigatus is a kind of common pathogen having a highpathogenicity. About 10 percent of endophthalmitis is caused by Aspergillusfumigatus.Almost all of Aspergillus fumigatus endophthalmitis would lead to blind.In the past it was considered that Aspergillus fumigatus endophthalmitis is merelycaused by extrinsic source,but recently Aspergillus fumigatus has been thinked highof as a endogenous pathogen along with the increase of the patients with lowimmune function. This type of fungus grows fast and has a strong pathogenicity. Itcan release a kind of resolvent protease which inhances the ability of growth andbreeding in the tissue.So once Aspergillus fumigatus endophthalmitis happens,pathogenetic condition develops fiercely.The pus filles up the eyeball and graduallypanophthalmitis happens,then fossa orbitalis is involved. In the end Aspergillusfumigatus endophthalmitis makes a worse damage of the tissue of eye and the visualfunction even results in dissemination all over the body.There is no unitive animal model of the fungal endophthalmitis currently. In myresearch by the pre-experiment and re-experiment,we choosed the Aspergillusfumigatus as the infection strain which acts as the common pathogen of Eurotiumand has strong virulence .When we choosed the guinea-pig with the weight of400-450g as fungal endophthalmitis animal model, we found that Aspergillusfumigatus albicans suspension with the perfect concentration of (1.0-1.5)x106(colony forming unit,CFU)/ml is very suitable. Thus we could obtain perfectfungal infectious symptoms of eye with no visceral and blood infection.It is the firsttime in the world that we established guinea-pig model of exogenous Aspergillusfumigatus endophthalmitis.Different from Monilia ,Aspergillus like to intrude into blood vessel. Usuallythe hypha will intrude in the vessel wall and grow on it.Big hypha can becomethrombus and block the vessel,which can lead to hemorrhage,ischemia and necrosisof the tissue.In my research through histopathological observation we foundAspergillus infected retina firstly. Hypha in the lumen of blood vessel of the retinaextend to the retinal extravascular tissue,which leaded to segregation of pigmentepithelium layer and Bruch membrane and promoted the detachment and necrosis ofthe involved retina following acute inflammatory reaction.The necrotic tissue andinflammatory cell soon formed the pus in the vitreous space.Because there is plentyof water and protein and no vessel in the vitreous body,it is easy for the pathogen tomultiply and cause inflammation and produce pus in it once infected.So we can findhypha in the pus.The guinea-pig model that we initiated to imitate exogenous Aspergillusfumigatus infection of the human eye has high coherence and repetition. There is nounitive animal model of the fungal endophthalmitis currently, and we initiated toestablish the guinea-pig model of Aspergillus fumigatus endophthalmitis to fill in theblank, which established the basis for the deep research of the fungalendophthalmitis.Fungal endophthalmitis is a kind of severe eye disease that can involve manysorts of tissue in the eyeball and take long time and make a worse damage of thetissue of eye and the visual function.So it is very important to early diagnosis. Nowthe laboratory diagnosis is mainly dependent on microscopy and culture ofmicroorganisms. The culture of microorganisms is always looking as the "goldstandard" in the laboratory diagnosis. But because of the little number of pathogensthat almost disperse in the whole vitreous cavity, the specimen which puncture fromvitreous cavity is so little that easily result in the sampling error and reducing thepositive rate of the cultures of fungi. And for the cultures take too long so we needto find a kind of sensitive and fast diagnostic method urgently.Polymerase chain reation (PCR),currently a new method of the moleculardiagnostic tool with a rapid development, contributes to microbiologic diagnosis ofthe little specimen ,or insensitive to the microscopy and culture for a long time, evento the pathogens which cann't be cultured. PCR diagnosis is could be used todetermine the conserved sequences of target genes universally and to make subtypetest by genes which have big discrepancies. It could not only do the specific testingfor one pathogen, but also do multianalysis for different species of fungi, bacteria orvirus. PCR techniques have much higher sensitivity and specificity compared withthe conventional immune method, and the testing time could also satisfy the clinicalneed, and it can rapidly establish the presence of even a few organisms (pg, ng) inthe specimen within 24 hours. Therefore PCR techniques will become the bestexperimental diagnosis method for rapid early diagnosis.First of all, how to choose the target gene is the key to PCR diagnosis.Mitochondria genes are the functional genes for cellular respiration and the survivalof eukaryocytes. The genes evolved in manner of maternal inheritance, having lessrecombination, and in favor of evolutionary researches for fungal molecular system.It is relatively easy to find the differences in species and enhance the sensitivity, asits evolutionary speed was faster than that of nucleus genes. Our researches choosegene of mitonchondria cytochrome b as the target gene, conducing to makeclassification of fungal infection, determination and rapid diagnosis. We chosed asegment of the noncoding region of fungal mitochondria cytochrome b gene as thetarget sequence, and design a pair of pan-primers to amplify the target gene segmentwith the length of 420bp or so. The primers are specific to the fungi and negative tothe DNA of bacterium(photosynthetic bacterium), human, animal and plant. Thetime of PCR to diagnose the fugal endophthalmitis was only about 8 hours, whichshowed the fungal pan-primer with highly specificity and PCR to quickly diagonosethe disease with the notable dominance. Secondly some factors in PCR reactioncould conduce false negative or false positive, such as the inhibitors in the aqueoushumor and vitreous humor, and parts of the inhibitors have thermal stability whichmakes heating useless to get rid of. The existence of such inhibitors could causefalse negative for PCR reaction. Our research took following approaches to extractthe target genes and remove inhibitors: SDS in the cell lysis solution was used tosplit the human cells, non-fungal pathogens, and the fungi with protease K. Then theDNA of pathogens was extracted by the continuous Tris-HCl.And the DNAdepositor was washed with the 70% cold alcohol, which the contaminantdisappeared. The next step of PCR would go on when the depositor was lysised bythe buffer after the dehydration. So we conclude to some extend the method of theextraction of DNA reduce the inhibitors and the occurrence of false-negativefindings, and increase the specific DNA production of the amplification and the rateof detection of the pathogens.It is an improvement of the sensitivity and specificity that was performed by thepurification of the DNA extraction and optimization of PCR reaction system.Especially we succeeded to validate the pan-fungal primers which we designed withcurrency and specificity.The specific and rapid diagnosis aiming at the fungalpathogen in the infected vitreous fluid of guinea-pig can help us solve many toughproblem clinically such as early etiological diagnosis and prompt , effectivetreatment of fungal endophthalitis.So this research has a significant prospect andvalue of clinical application.The principle for the treatment of endophthalmitis is to reduce the concentrationof intraocular organisms rapidly and promote the diffused distribution of antibioticsin the vitreous. The barriers of retinal capillary endothelium and the tight junctionsof pigment epithelium block the drug in the blood and subconjunctiva frominfiltrating into ocular posterior segment. Even enough use of amphotericin B takenintravenously or subconjunctivally could not reach the effective anti-infectiousconcentration in the vitreous, so the inflammation could not be controlled radically.In order to reach the required concentration and reduce the intraocular concentrationof pathogen, the solely reliable method is to inject the drug to the vitreous chamberdirectly.,which pass the blood-retinal barrier and can incease the drug concentrationin the infected position promply.Since the last century,intravitreal amphotericin Bwith dosage of 5μg~10μg is widely chosen to treat fungal endophthalitis.Amphotericin B Liposome(L-AmB) is a kind of new preparation of Amphotericin B.However as recently the occurrence of some drug resistance strains and serioustoxic effect and side-effect ,it restrained the widespread application of AmphotericinB.It is a great beginning to the anti-fugal drugs that the manufacture ofAmphotericin B Liposome is so extremely effective that it increases the effection oftraditional Amphotericin B and decreases the toxicity(especially the renal toxicity),especially for the treatment of the patients who are hard to tolerance the traditionalAmphotericin B or the poor effection with the fugal endophthalmitis.Theinvestigation detected when we suspected the the fugal endophthalmitis, theeffective drug concentration should be achieved quickly in the eyeball before theserious destruction of the tissue and the thalla were not yet mass multiplied.Theapplication of the anti-fungal drug treatment early ,whether association withvitrectiomy or not, would obtain the better therapeutic efficacy. In our experimentthrough intravitreal injection of L-AmB with different dosage treating Aspergillusfumigatus endophthalitis in the initial stage(24 hours after infection),we find thatL-AmB with the concentration of 20μg /0.02ml is therapeutically effective and safefor retina and the other tissues in the eyeball. We evaluated the influence of L-AmBto the retinal function by ERG.. The result was that the amplitude of wave b of ERGwas not obviously descent after the drug injection with 20μg /0.02ml L-AmB.Andthe retinal structure was regular by histology examination. The pigment epitheliumof retina was integrated ,the cells structure lined up in order, and there were no cellsdeletion and necrosis through the transmission electron microscope. So it wasproved there was no toxic effect of 20μg /0.02ml L-AmB at the level of organs,tissues and cells. It is significant for us to choose intravitreal injected L-AmB to treatAspergillus fumigatus endophthalitis and prevent unhealing and relapse. At the sametime this method develops a new, safe, effective therapic way.