| BackgroundHIV testing is the basis and indispensable technical support for AIDS prevention and treatment work,only as early as possible diagnose to find HIV infection,early treatment,can we to reduce HIV transmission and extending the life of an infected person.However,the current conventional HIV testing modes in China cannot cover the whole infected population.There are still about one third of HIV infected people or AIDS patients have not been tested.And the proportion of newly infected people is low,that is,the proportion of late infected is high.Therefore,our research team have searched out an innovative HIV testing mode of self-collected samples for HIV testing(SCT),which is a kind of self-help to collect samples in the privacy space and mail them to a professional laboratory for HIV testing,obtain test results and services anonymously using the Internet,based on the policy"China’s 13th five-year plan of action to curb and combat AIDS"to encourage"explore HIV self-detection ways by the pharmacy,network sales test reagents and other methods",platforms of the extensive use of Internet and the perfect HIV testing laboratory network in China.Previous studies have proved that SCT is a feasible and acceptable HIV testing mode,which based on urine sample self-collection.However,there are some deficiencies of urine samples,including it can only be used for HIV antibody screening test,less sensitive and specific than blood samples,and there are significant difference among different populations.Dry Blood Spots(DBS)as a kind of blood sample easy to collect and transport,which have been used for individual and remote areas HIV testing in American since 1996.DBS not only can be used for the HIV antibody and antigen detection,but also can be used to test the HIV nucleic acid,it is with more than 99%consistency with the matched plasma samples.But there is no standard operating procedure and commercial reagent for HIV antibody detection in DBS in China currently.In this study,we are first to extended applied SCT mode based on urine samples in order to do verification and evaluation.Then,a variety of effective and convenient methods for HIV antibody testing in DBS have been systematically optimized using plasma HIV antibody diagnostic reagents,based on the preliminary research foundation of our research team and the current domestic HIV laboratory antibody testing status.Finally,we to explore application effect of the SCT innovative mode with DBS self-sampling.This study aims to research and provide the technical support and scientific basis for the clinical application of DBS self-collecting and mailing during HIV detection innovation mode(SCT)in China.In order to provide a more complete,accurate and efficient innovative HIV detection strategy,further to strengthen the AIDS detection and prevention work in China.Objetives1.To verify the urine self-collection for HIV testing mode application effectiveness.To evaluate and verify the acceptability and applicability of this HIV testing mode(SCT),by clinical applied the urine self-sampling and mailing,through calculated the positive rate of HIV infection,the rate of new find infections,the result search rate and the participants’preferences for various patterns.In addition,we evaluated the benefits of this HIV testing mode SCT by comparing with other traditional testing modes.2.To resaerch HIV antibody test methods based DBS.A scientific and systematic research was conducted to establish a variety of HIV antibody detection methods besed DBS samples.Systematic and scientific researched the uniformity and stability of DBS samples,when used in the DBS self-collection for HIV testing innovative mode.Work out the standard operating procedure of HIV antibody testing based DBS,which provided the scientific basis for clinical application.3.To verify the DBS self-collection for innovative HIV testing clinical application.To evaluate and verify the acceptability and applicability of the innovative HIV testing mode based on DBS self-collection,through calculated the positive rate of HIV infection,the rate of new find infections and the recent infection rate,the result search rate and the testing accuracy,etc.In order to explore a more complete,accurate and efficient innovative HIV testing mode,which is could strengthen the extension of HIV testing in China in future.Methods1 To verify the urine self-collection for HIV testing mode application effectiveness.From June 2016 to January 2017,pharmacy distribution form,grassroots organization mobilization form,pilot universities form by free distribution and self-service machine sales methods were applied.Analysis data from service package distribution,sample collection and delivery,laboratory sample acceptance and test,result search online and follow-up,then respectively calculated the number of service packages distributed or purchased,laboratory urine sample recovery rate and sample qualification rate,positive detection rate,result search rate online and search times of negative and positive people,follow-up rate of positive person,new diagnosis rate and rate of receiving antiviral treatment with each form.Finally,evaluate the benefits of SCT mode by comparing it with other traditional testing modes.2.To resaerch HIV antibody test methods based DBS.Using HIV positive and negative blood and domestic widely used HIV antibody diagnostic kit(ELISA)to optimize elution conditions of DBS samples,including temperature(room temperature 24±2 ℃ and 4 ℃),elution liquid composition(PBS,PBS containing 0.5 ‰ Tween20,PBS containing 1.0 ‰ Tween20),elution liquid volume(300μl,400μl,50μl),the elution time(2 hours,4 hours,6 hours,12 hours,24 hours).Furthermore,the DBS elution volume and sample addition volume suitable for the colloidal gold immunochromatographic method and western blot method for HIV antibody diagnostic kits optimized.Reagent A and reagent B from two different manufacturers were used in parallel for each of above methods.The sensitivity and specificity of the each optimized method were verified by using 189 HIV-positive and 200 HIV-negative DBS samples paired with plasma.Additionally,using the DBS-plasma matched samples(three serial dilutions and 71 clinical samples)to optimize the elution liquid volume(500μl,600μl,800μl,1000μ1)of LAg-Avidity EIA method for HIV-1 recent infection detection of DBS.Finally,systematic researched stability of the DBS samples in different conditions(seal without desiccant,seal with desiccant,DBS elution liquid),different temperature environment(24 ±2℃,37℃,4℃,-20℃)based on above HIV antibody detection methods3.To verify the DBS self-collection for innovative HIV testing clinical application.From June 2017 to September 2018,the innovative SCT mode based on DBS self-collection was clinically applied to MSM population in Beijing,network application population in some regions,and population from pilot VCT in some regions.Follow up or HIV nucleic acid test results were used as the infection criteria.We calculated the detection accuracy,true positive detection rate,true negative detection rate,missed detection rate and false positive rate of each form to evaluate methodological.Calculated the number of laboratory DBS sample recovery and sample qualification rate,positive detection rate,online result query rate of positive subjects,follow-up rate of positive subjects,new diagnosis rate,etc.to conducted the SCT mode based DBS samples verificationResults1 To verify the urine self-collection for HIV testing mode application effectiveness.Firstly,the urine samples recovery rates of pharmacy distribution form,grassroots organization mobilization form,pilot university form were 86.0%(430/500),63.9%(1977/3092)and 39.49%(378/957),respectively.Among them,the urine samples recovery rates of free distributed and sale by vending machine ways in pilot university form were 33.0%(251/760)and 64.5%(127/197).Secondly,the sample qualification rates of the three forms were 100%(430/430),96.7%(1911/1977)and 99.5%(376/378),respectively.Among them,the HIV antibody positive detection rate in drugstore distribution form was 16.3%(70/430),significantly higher than grassroots organization mobilization form(7.59%(145/1911)and pilot university form(1.86%(7/376).Thirdly,more than 80%of the HIV positive people actively searched the test results,and the maximum number of search times for HIV positive and negative people were 16 and 31.Fourthly,the follow-up rate of the HIV positive person in the university model is the lowest(14.30%),and the other two models are above 64%.The blood confirmation rate of positive urine screening was 67.40%and the new diagnosis rate of positive was above 93.1%.The positive detection rate of SCT mode is 100-200 times higher than conventional mode(0.08%).2.To resaerch HIV antibody test methods based DBS.The consistency between DBS and plasma samples of ELISA was 99.49%(387/389)-99.74%(388/389),and the Kappa value was 0.990 and 0.995(P<0.001).The consistency between DBS and plasma samples of colloidal gold immunochromatography was 98.39%and the Kappa value was 0.964(P<0.001).The consistency between DBS and plasma samples of immunoblotting was 99.03%(307/310)and the Kappa value was 0.979(P<0.001).Testing correlation coefficient between DBS and plasma samples of LAg-Avidity EIA for HIV-1 recent infection detection was 0.955-0.968(R’=0.912,R’=0.936).DBS sample stability is related to reagent performance and the OD value increased after place at 24+2 C for 8 weeks,37 C for 4 weeks and 4’C for 8 weeks.With the placed time extension at 24±2℃and 37 C,the RIBA bands of positive samples’weakening and negative samples appearing interference bands with time prolongs and increasing humidity.There was not change significantly after repeated freeze-thaw for 6 times at-20℃.3.To verify the DBS self-collection for innovative HIV testing clinical application.3.1 Methodology verification:true positive detection rates for ELISA and colloidal gold assays were 100%(218/218)and 92.66%(202/218),and true negative detection rate were 98.57%(1861/1888)and 100%(1888/1888).There were 1.43%(27/1888)false positive rate of ELISA and 7.43%(16/218)false negative rate of colloidal.Confirmation accuracy of recombinant western blot for retest screening HIV positive samples was 100%(218/218).3.2 Verification of mode application:a total of 2107 DBS samples which prepared by the active participants were received and the sample qualification rate was 99.95%(2106/2107).The HIV antibody positive rate of innovative SCT mode based DBS-collection was 10.35%(218/2106),and the the HIV antibody negative but HIV DNA poaitive that acute infection rate was 0.47%(10/2106).The highest HIV antibody positive rate 17.65%(33/187)was online applicants,these rate of MSM population and population from pilot VCT were 9.97%(130/1304)and 8.94%(55/615),respectively.Follow-up confirmation or HIV DNA retest results as criteria for infection.HIV antibody positive testing precision rate was 100%(218/218)of innovative SCT mode based DBS.Among the participants,the result search rates of were 76.8%-100%,and the highest number of search times were 57.Among the MSM people in Beijing,the new diagnosis rate of positive people was 81.81%(108/132),and the HIV antibody positive detection rates of the rapid blood test onsite mode and SCT were 9.05%(118/1304)and 9.97(130/1304),respectively.SCT based DBS could excluded 47.5%(29/61)of HIV antibody false positive during self-test for the reagent purchased online.ConclusionsIn this study,through the verification and evaluation of the innovative SCT mode of urine self-collection,research the standard method for HIV antibody detection with DBS samples,and the applition of innovative SCT mode based on DBS self-collection.It is shown that:1.The urine self-collection for HIV testing mode has a good feasibility and population acceptability,and the more than 10%positive rate significant higher than conventional HIV testing mode.2.DBS samples have good accuracy and stability used for HIV antibody detection,can be successfully used in innovative SCT mode with DBS self-collection.3.Not only the innovative SCT model based on DBS self-collection has a good feasibility and population acceptance,but also it effectively test the insidious infection that does not want to be exposed and can effectively make up for the limitation of urine sample which had been used SCT mode.4.This study has proved that the innovative SCT mode could facilitate find more new diagnosis,recent and acute infections.5.This study provides a convenient,feasible and effective innovative path to expand HIV testing and monitoring insidious infected people in resource constrained and remote areas.6.This study provides ideas and scientific basis for HIV antibody detection reagents manufacturers to develop reagents used for DBS in China. |
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