The Epidemiology Of SLE Kidney And Detection Of SLE With Deep Fungal Infection By Multiplex Fluorescent Quantitative PCR

Ⅰ.The Purpose and Meaning of ResearchSystemic lupus erythematosus（SLE）,a common disease of autoimmunity,can cause damage to the internal organs of human body.Moreover,the damage caused to kidney is the commonest disease,which is called lupus nephritis（LN）.The type of LN can only be affirmed through pathological examination with the help of kidney puncture.However,this kind of examination is traumatic,and it also depends on stipulations,such as the state of patient and medical conditions,thus not all of the LN patients can take the pathological examination of kidney.Therefore,we intend to initially figure out the pathological type of kidneys of LN patients via the clinical manifestation and the results of some non-traumatic examination,so that we can provide some certain basis for the treatment of the disease and lessen the patients’ pain.Further more,as to the pathogenesis of the kidney damage of lupus patients, lupus nephritis is a chronic autoimmunity disease,which possesses various kinds of auto antigens and induces many kinds of autoantibody.Therefore,the composite induced by LN has multiple characters.There are three kinds of immune globulins, namely,IgG,IgA and IgM in the composite,and the intensity of deposit is comparatively strong.Activating Complement through classical pathway is one characteristic of it,however,Clq is a subunit of C1 and the initial element of the activation of classical pathway.The recent research shows that Clq has something to do with the paroxysm of SLE,and the research of the relationship between them is based on the examination of blood,as a result,it can not directly reflect how deposit is related to the Clq of kidney and the disease.What’s more,the study on this aspect is not reported yet.Therefore,by means of examination of kidney pathology,we intend to get to know the relationship of Clq and SLE so as to provide basis for relationship between Clq and the pathogenesis of SLE.In the treatment of SLE and LN,in order to better the state of illness,not only is the hormone regularly used,but also immunosuppressant or cytotoxic drugs are added,so Cyclophosphamide（CTX） and Mycophenolate Mofetil（MMF） are typical medicines.In clinical operation,CTX is a traditional medicine for treating LN,and it is inexpensive and comparatively effective.It has positive effects on relieving the patient’s ill state,repairing the damage of kidney,and improving the patient’s life quality,however,its side-effect is comparatively more and heavier,which limited the application of CTX.With the increasing use of MMF,the treatment of LN is in some ways improved.According to many reports,it is better than CTX in treatment effects, and the side-effect of it is less frequent and less serious.However,according to the observation of our clinical operation,the treatment effect of MMF,compared to CTX, is not as good as what has been reported.In order to further understand the curative effect and safety of these two medicines,we apply the evidence-based medicine （EBM）,which is mostly advocated in the clinical treatment of the disease throughout the world.Under the guidance of EBM,we select the method of Meta analysis to carry out the comparison and assessment of the validity and safety of MMF and CTX in treating LN.Meta analysis is a quantitative and comprehensive analysis on many researches which are identical in purpose and close in characters.It does not conform to a simple method of statistics,but involves a series of procedures,such as raising problems,establishing standards of including and excluding,searching related researches,gathering basic information,comprehensive analyzing and reporting the results.On the selection of LN type,we set the diffuse proliferative lupus nephritis （DPLN） as research orientation,not only because DPLN one of the clinically commonest type of LN,but also the Meta analysis of the treatment of DPLN is not reported yet,which makes it representative and innovative.Nowadays,the constant development of medicine and technology of treating SLE patients plays an important role of improving the survival rate and life quality of SLE patients.However,the poor immunity of SLE patients is an undeniable fact,to make matters worse,the application of hormone and immunosuppressant（like MMF and CTX we have mentioned）,as well as new technologies like the implantation of autologous hemopoietic stem cell transplantation（AHSCT） which requires a great amount of CTX or other medicines,can make the lupus patients’ immunity poorer and thus more liable to combine the infection of various pathogenic microorganism, such as virus,bacteria,fungi,etc..When a lupus patient combines the infections of deep fungus,the symptom is not typical and will cause the confusion of treatment. Whatsoever,the diagnosis should be achieved by the fungal culture.As sometimes the patient is likely to combine more than one kind of deep fungal infection,it takes a long time to diagnose,and it’s impossible to quantitatively analyze the degree of infection.As a result,we try to find out a new technology,requiring shorter time, quantitative,and capable of examining many types of fungi simultaneously,so that it can facilitate us achieve the diagnosis of the SLE patients’ combining of deep fungal infection.Multiplex fluorescent quantitative PCR is a newly popular diagnosing technology.Its advantage is that,by the design of multiple probes and the examination of one specimen,it can examine different types of microbes at the same time.This examining technology can display the results in a few minute and monitor the infectious degree quantitatively,which is very favorable to the judgment of the seriousness of disease.At present there is no report on this technology examining the SLE patients’ combining deep fungal infection.Should this technology be successful, it would achieve the above mentioned purpose and bring an important breakthrough to the clinical treatment.To carry out the research,We choose the common deep fungus,candida albicans and aspergillus flavus to be monitoring objects.Ⅱ.The Method and Content of Research1 Reseach on the Clinical Characteristics of SLE,Pathology of Kidney and Their Relationship with Clq1.1 Choose SLE patients taking the examination of light and immunofluorescens microscopy pathological examination with the help of kidney puncture.Divide these patients into three groups,namely,Group anteriorⅣ、GroupⅣand GroupⅤ. Observe the clinical manifestation and lab examination of these SLE patients in each group so as to study the relationship between the clinical manifestation of SLE and the Pathology of Kidney1.2 According to the pathological result,divide these patients further into Clq positive group and Clq negative group so as to study how the clinical manifestation and kidney pathology are related to Clq.2 Meta Analysis on MMF and CTX Treating DPLNSearch the databases such as PUBMED,China National Knowledge Infrastructure（CNKI）,and Chinese Biomedical Database（CBM） via computer,and manually search 16 kinds of medical Journals such as Nephrology,Rheumatology, Dermatology,etc.,in order to collect the random experiments of comparing MMF and CTX on treating DPLN.Through Meta analysis,assess several aspects of treatment,including effectiveness,recurrence rate,death rate,24-h Urine protein, level of serum creatinine,activity index of disease,chronic index of disease,infection, decrease of leukocyte,amenorrhea,and diarrhea,and assess the effectivity and safty.3.Research on the examination of SLE combining deep fungal infection by multiplex fluorescent quantitative PCRAccording to the gene sequences of candida albicans and aspergillus flavus through American Type Culture Collection（atcc）,design two pairs of primers and Taqman probes with the aid of molecular biology software,and optimize the conditions of multiplex fluorescent real-time PCR reaction.Assess the positive rate of MFQ-PCR through 20 specimen of SLE combining deep fungunal infection,the sensitivity of MFQ-PCR by examining 20 Suspected SLE combining deep fungal infection,and specificity through detecting 20 non-candida albicans and non-aspergillus flavus.Ⅲ.The result of research1 Reseach on the Clinical Characteristics of SLE,Pathology of Kidney and Their Relationship with Clq1.1 The Relationship of Clinical Characteristics of SLE and Pathology of KidneyAmong the SLE patients of different types of renal pathology,it is statistically meaningful（p＜0.05） to examine the incidence of disease such as arhralgia,butterfly erythema,light sensitivity,hypertension,anti-ds-DNA,decrease of HGB and complement,increase of BUN and Cr,haematuria,Urine protein（3+）,24-h Urine protein（＞0.5）,urine C₃,nrineα-M,nrine Lys,urine NAG,and positive Clq;1.2 The Clinical Characteristics of SLE,Pathology of Kidney and Their Relationship with Clq posivtive1.2.1 Involving the renal pathology in the logist regressive research We get the regression equation--LogitPl=0.777+1.884X1+1.630X2（X1 and X2 stand for type-ⅣLN and diarrhea respectively,and their OR values are 6.582 and 5.102）; correlation coefficient of equation:R²=0.521.The percentage correct of the equation is 76.7%,and its sensitivity is 94.8%;1.2.2 Not involving the renal pathology in the logist regressive research We get the regressive equation--LogitP2=3.296-1.667X1+1.425X2+1.209X3（X1～X3 stand for gender,diarrhea and C₄ decrease respectively and their OR values are 0.189、4.160 and 3.350）;correlation coefficient of equation:R²=0.471.The percentage correct of the equation is 80.7%,and its sensitivity is 90.5%.2 Meta Analysis on MMF and CTX Treating DPLN10 pieces of documents in total are used as reference in this research.There is no obvious difference between MMF group and CTX group in the effect of treating DPLN patients,such as the relief of ill state,illness recurrence,death condition, 24-hour Urine protein and serum creatinine decrease（March,June and December）. However,MMF group brings out better effect than CTX group does when improving AI index,furthermore,in MMF group,the chance of infection,decrease of leukocyte, amenorrhea is less than that of CTX group,which has statistic meaning（P＜0.05）.3.Research on the examination of SLE Combining deep fungal infection by multiplex fluorescent quantitvative PCRAccording to multiple fluorescence quantitative PCR examination,both of the positive rate and specifity of SLE combining candida albicans and aspergillus flavus are 100%;the sensivtity of multiple fluorescence quantitative PCR method and that of fugus culture method are 75.00%and 40.00%respectively,thus,the comparison of the sensitivities of the two methods reveals statistic difference（P＜0.05）.Ⅳ.The Conclusion of ResearchAs is revealed in the first part of this research,the clinical characteristics of SLE patients are in some ways distinguishable,because their kidneys are of different pathological types.Through the clinical manifestation and the non-traumatic examination,we can initially judge the SLE patients’ pathological type of kidney damage As to the research of the Clq deposit in the renal tissue,we discovered that we can preliminarily figure out the possibility of Clq depositing in kidney by resorting to the two equations seperately（depending on whether take pathological examination of kidney or not）,which brings great convenience to the clinical doctors and in some ways lessens the pains of patients.Moreover,we discovery another phenomemon,namely,diarrhea is included in the two equations as independent variable and it is also dangerous element,which means greater possibility of Clq deposting in kidney to the LN patients with diarrhea.The specific causes of this phenomenon require our further research.According to EBM in the second part,we find that MMF is much safer than CTX. On treating DPLN,MMF can not only improve the AI index（which has been reported in recent documents）,and MMF group also brings out better results than CTX group does while most of the treatment index are quite similar.Such research result is of great importance for us to clinically treat SLE.During the third part of the research,we find that the system of multiplex fluorescent real-time PCR based on Taqman probe technology,which is fast,sensitive, specific and quantifiable,can simultaneously examine SLE patients’ combining infection of candida albicans and aspergillus flavus.If this technology can be applied to clinical diagnosis,it will be a significant breakthrough for the diagnosis of fungi and reduce the financial cost of patients greatly.What’s more,this technology can inspire us to carry out further research,that is to say,to raise the number of probes so that we can achieve that the simultaneous and quantitative examination of bacteria, virus and fungi,which shall be a great thrust for the clinical diagnosis.