The Design And Preliminary Application Of Patch Testing Of Patients With Dapsone Hypersensitivity Syndrome

Background:Dapsone,(DDS),containing antibacterial and anti-inflammatory effects,is an essential medicine to the treatment of leprosy and is widely used to the treatment of malaria,pneumocystiscarinii pneumonia(PCP)caused by HIV infection and many chronic inflammatory diseases.Chemically,dapsone is an aniline derivative.As a sulfone,it shows the structure of a sulphur atom linking to two carbon atoms.Solubility of dapsone varies over a wide range depending on the solvent used(e.g.water,0.2 mg/mL vs.methanol,52 mg/mL).Following oral administration,dapsone is almost completely absorbed from the gut with bioavailability exceeding 86 %.Peak serum concentrations are attained within 2–8 h.After ingestion of a single 50–300 mg dose of dapsone,maximum serum concentrations range from 0.63 to 4.82 mg/L.Under steady-state conditions,100 mg/day(the dose most frequently used)results in serum concentrations of 3.26 mg/L(maximum)and 1.95 mg/L(after 24 h).These dapsone serum concentrations attained in vivo must be kept in mind when interpreting the results of in vitro investigations(see below).After absorption,dapsone undergoes enterohepatic circulation.It is metabolized by the liver but also by activated polymorphonuclear leukocytes(PMN)and mononuclear cells.In the liver,dapsone is metabolized primarily through acetylation by N-acetyltransferase to monoacteyldapsone(MADDS),and through hydroxylation by cytochrome P-450 enzymes,resulting in the generation of dapsone hydroxylamine(DDS-NOH).The biggest side effect of dapsone is a serious drug hypersensitivity syndrome appearing on some patients after taking medication.Its clinical manifestations are sudden onset of papule or exfoliative dermatitis with fever,enlarged lymph nodes,mononucleosis and liver or impairment of hematopoietic system.It usually occurs after taking dapsone for 4 to 6 weeks,so it is also called” 5-week rash”.Its incidence rate is 0.5% to 3.6% and death rate 9.9%.The adverse reactions of this drug was officially named dapsone syndrome in 1951.At present,the diagnosis of this syndrome is mainly based on the diagnostic criteria put forward by Richardus and Smith in 1989.The diagnosis can be confirmed when the patient develop two or more manifestations including rash,fever,abnormal liver function and enlarged lymph nodes after taking this medicine.However,the above diagnosis is lack of valid laboratorial diagnostic index and specificity.Hence it is difficult to differentiate it from the infection and inflammation emerging from the disease itself.As for the treatment of the disease,the patient is likely to take various drugs at the same time and leads to difficult judgment and the accurate time to stop medication.As a result,missed diagnosis and error diagnosis are severe clinically.DDS syndrome is a T lymphocyte mediated by a drug hypersensitivity,in the body for the first time exposure to sensitizing drugs,the body T lymphocytes are activated sensitization.When the body re-exposure to the drug,sensitized T lymphocyte activation and proliferation,the release of cytokines IFN-?,etc.,to attract and activate macrophages,in the local production of T cells and macrophages infiltration-based inflammatory response.Patch test as a method of determining the body allergic diagnosis method is based on the above principles,so far in the dermatology application has been 120 years of history.At present the international diagnosis of clinical common 28 kinds of sensitizing drugs(carbamazepine,ibuprofen,amoxicillin,diclofenac sodium,etc.)has developed an independent intellectual property rights patch kit,has been the US FDA,China cFDA and EU certification,widely used in clinical,drug-induced adverse reactions to the diagnosis and differential diagnosis provides an effective tool to obtain a good social and economic benefits.Objective:To develop dapsone reagent,establish the immunization confirmatory methods of dapsone syndrome and provide effective techniques for the diagnosis of this disease.Research Object:1.Dapsone syndrome group: 24 cases of lepers clinically diagnosed with dapsone syndrome and carrying HLA-B*1301.2.Control Group: 25 cases of lepers with HLA-B*1301 who had taken dapsone but without dapsone syndrome.50 healthy volunteers.Inclusion Standard:Disease Group:1.New lepers who had taken dapsone from January,2008 to December,2016;2.Patients taking dapone with suspected dapsone syndrome,such as fever,rash,abnormal lung function,lymphadencectasis during the treatment,which was consistent with the diagnostic criteria proposed by Richard and Smith;3.Patients carrying HLA-B*1301 alleles;4.Patients who agreed to participate in this research and signed the written informconsent.Control Group:1.From January,2008 to December,2016,new lepers who had taken dapsone for more than 1 month;2.Patients taking dapone without fever,rash,abnormal lung function,lymphadencectasis symptom during the treatment;3.Patients carrying HLA-B*1301 alleles;4.Patients who agreed to participate in this research and signed the written inform consent.Exclusion Standard:1.Patients allergic to sulfanilamide,sulfonamides,furosemide,thiazide,sulfonylurea and carbonic anhydrase etc.;2.Patients who cannot provide the accurate information;3.Patients with serious diseases,such as heart failure,malignancy.Methods: The research is divided into three parts.Research Contents: This research can be divided into 3 parts:Research I: develop dapsone reagent for clinical application,explore stable reagent matrix and concentration range,and check its stability: a case with suspected dapsone syndrome as well as positive clinical diagnosis and provocation test was taken as a research object,to explore appropriate substrate and concentration range of dapsone reagent;Research II: carry out content determination on the prepared reagent and check its stability.Research III: check the sensitivity and specificity of reagent: reagent was adopted to test 23 clinically suspected dapsone patients with HLA-B*1301 and 25 lepers with HLA-B*1301 who had taken dapsone but didn't have suspected dapsone.50 healthy volunteers were taken to exclude the skin irritation caused by the patch reagent itself.Chi square test was used to confirm the statistical significance of the differences among the groups,and to confirm the sensitivity and specificity of the clinical application of the reagents.Research Methods:Research I: Dissolution method was adopted to observe the dissolution degree of vaseline basis,polyethylene glycol 200 matrix,polyethylene glycol 200 matrix +dimethyl sulfoxide,polyethylene glycol matrix+oleic acid,polyethylene glycolmatrix+azone in dapsone,and to adjust the concentration of photopatch reagent according to the patch test results.Research II: High performance liquid chromatograph was adopted to determine the content of prepared Vaseline,and the photopatch reagent of polyethylene glycol 200 matrix,and in the meantime,to investigate its preliminary stability.Research III: Dapsone patch test was adopted on the research objects,and the method was as follows:1.Put the dapsone patch reagent in place according to the concentration of 0.1%,0.5%,1%,5%,10%,15%,20%,and 25%.2.Tear the patch instrument from the triangle corner,add dapsone patch reagent and blank control successively into the patch instrument,write down the concentration order of photopatch regent.3.Fully expose the patient's back skin,select normal skin without rash or hyperpigmentation for experiment.4.Disinfect the skin with alcohol wipes,paste the patch tester firmly on the patient's back,press it to empty air.5.Mark the four corners of the testers with marker,and write down the data on the experiment record.Special attention should be given to the position of patch reagent of different concentration.6.Ask the patient to tear off the tester after 48 hours,during that time they should not take a shower,do farm work,strenuous exercise,drink,etc.The tester should be tear off once severe reaction occurs during the test.7.After 72 hours,or after 24 hours of tearing the tester,observe the test results.Clean the tested part with alcohol wipes,mark the four corners of the tester with marker,observe whether the tested part has red spot,papule,or bubble(the experiment results are not significant in some patients,detailed observation is needed).According to the International Contact Dermatitis Research Group,the patch test result must be determined by the same technician.Statistic method:The analysis of research results was conducted in ITT(intention to treat)groups,which indicates all the participants of the research who had taken medicine for at least one time.SPSS 16.0 was adopted in data statistics and application to check whether every index difference between experimental group and control group was of great significance according to the chi-square of test research purposes and information.All the statisticaltests were two-sided tests.The test level was defined as?=0.05.If P value was less than0.05,it would be considered that the difference was of statistical significance.Research Results:Research I: Based on the comparison among dapsone vaseline matrix,polyethylene glycol 200 matrix,polyethylene glycol 200 matrix+dimethyl sulfoxide,polyethylene glycol matrix+oleinic acid;polyethylene glycol matrix + azone,polyethylene glycol 200 matrix + dimethyl sulfoxide was finally selected as the best preparations.Research II: Polyethylene glycol 200 was adopted as the photopatch of matrix.It was stored under normal temperature status and 4? low temperature for 6 months,but there was no markedly declining tendency in sample content,and low temperature storage and normal temperature storage exerted no difference on the sample stability.Such matrix can guarantee that the sample can maintain stable state within certain time,with no need to prepare when it is needed.Research III: Patch test was conducted on 23 cases of patients with dapsone syndrome nationwide,among which 10 cases were positive and 13 cases negative.The 25 cases of patient who had taken dapsone but didn't have dapsone syndrome were all negative in patch test.50 healthy volunteers were negative in dapsone test.Conclusion: This research sets a method to confirm dapsone syndrome immunity based on patch test,providing an effective laboratory technique to the accurate diagnosis of this disease.It also offers a new thought and reference to confirm the immunity of hypersensitivity syndrome caused by other drugs.Therefore,it is of vital significance in judging patient's allergic drugs and confirming the disease diagnosis in clinical practice.