| BackgroundIn order to prevent and control the spread of HIV(Human immunodeficiency virus,HIV),HCV(Hepatitis C virus,HCV),and TP(Treponema Pallidum,TP)scientifically and effectively,and to know about the trends of HIV,HCV,and TP,China has established National AIDS sentinel surveillance system.So far,our country has established nearly 1975 sentinel points throughout the country,and hundreds of thousands of tests need to be tested every year,including HIV,HCV,and TP.At present,the method of sentinel sample detection in China is to collect plasma,and then transport it to the laboratory by special personnel.Each sample is tested for HIV,HCV and TP respectively.Meanwhile,the positive plasma samples need to be stored for a long time.However,these detection systems still face great challenges in the actual work.There are many problems,such as a large number of manpower needed to collect plasma samples on site,high requirements for sample transportation,especially high-level biosafety requirements for HIV highly pathogenic microorganisms,large storage space for long-term accumulation of positive plasma samples,and inability to trace to individuals using plasma samples when traceability is needed.Compared with plasma,dried blood/plasma spots samples(DBS/DPS)have many advantages,such as good stability,small space occupation,small blood volume,simple storage,no need of cold chain transportation,and minimizing the risk of biological hazards.Previous studies have proved that DBS are an effective method for monitoring genetic diseases.DBS is helpful to solve various problems in collection,transportation,preservation and traceability.However,there is no relevant research on whether DBS to alternative to plasma can be applied to HIV sentinel monitoring and whether it can combine with China’s HIV sentinel monitoring system,in order to improve the storage,transportation,biosafety and traceability of samples in China’s HIV sentinel detection/monitoring work.It is of great practical value to study and establish a more suitable laboratory detection method for sentinel surveillance in China,so as to improve the overall efficiency of sentinel surveillance in China.Methods1.Establishment,evaluation and strategy on the serological method for simultaneously screening for HIV,HCV and TP infection based on one DBSExperimental parameters on optimal experiment condition of DBS samples were optimized,such as volume of blood,size of DBS sample,volume of eluent,elution buffer and duration and temperature of elution.Lab basic panel,Lab analytical specificity panel and Lab precision panel were constructed to be used for evaluating the linear range,sensitivity,specificity,precision and anti-jamming capacity for the DBS/DPS-based ELISA method established and the stability of DBS/DPS was evaluated as well.A total of 429 paired plasma and DBS samples were collected from MSM and PWIDs in Heilongjiang Province and Jiangxi Province between June 2018 and October 2019.The seroprevalence of HIV,HCV,and TP was determined based on the plasma test results.The clinical sensitivity,clinical specificity,positive predictive value(PPV),and negative predictive value(NPV)of the DBS/DPS-based assay were calculated for the detection of the 3 analytes.The consistency coefficient Kappa was calculated for the 3 analytes between the plasma and DBS/DPS method.Develop DBS antibody screening strategy and evaluate the application of these strategies using laboratory routine testing results as reference standard.DBS antibody screening strategy is to screen for anti-HIV,anti-HCV and anti-TP using one DBS.2.Establishment and evaluation on the serological method for simultaneously screening for HIV,HCV and TP infection based on one DPSExperimental parameters on optimal experiment condition of DPS samples were optimized,such as volume of plasma,size of DPS sample,volume of eluent,elution buffer and duration and temperature of elution.Lab basic panel,Lab analytical specificity panel and Lab precision panel were constructed to be used for evaluating the linear range,sensitivity,specificity,precision and anti-jamming capacity for the ELISA method established using one DPS and the stability of DPS was evaluated as well.A total of 329 paired plasma and DPS samples were collected from MSM and PWIDs in Heilongjiang Province and Jiangxi Province between June 2018 and October 2019.The seroprevalence of HIV,HCV,and TP was determined based on the plasma test results.The clinical sensitivity,clinical specificity,positive predictive value(PPV),and negative predictive value(NPV)of the DBS/DPS-based assay were calculated for the detection of the 3 analytes.The consistency coefficient Kappa was calculated for the 3 analytes between the plasma and DBS/DPS method.The detection performance of one DBS and one DPS ELISA methods were compared and analysed.3.The study of Establishment and strategy on the molecular method for simultaneously screening for HIV,HCV and TP infection based on one DBSBased on the research of nucleic acid extraction of DBS samples in our laboratory,further optimize and improve the to meet the needs of simultaneous screening of HIV,HCV and TP nucleic acids,in order to develop a molecular method for simultaneously screening for HIV,HCV and TP infection based on one DBS.Develop strategy of the combination of one DBS ELISA molecular method and evaluate the application of these strategies using laboratory routine testing results as reference standard.strategy of the combination of one DBS ELISA molecular method is that the first step is to screen for anti-HIV,anti-HCV and anti-TP using one DBS ELISA method;the second step is that HIV-1 RNA or HIV-1 DNA testing is performed in anti-HIV positive samples,HCV RNA testing is performed in anti-HCV positive samples and DBS-based ELISA testing is performed in anti-TP positive samples;the results is based on the results of the second-step.Results1.Establishment,evaluation and application strategy on the serological method for simultaneously screening for HIV,HCV and TP infection based on one DBSThe optimized assay conditions were:blood volume,70-100 μL;DBS size,whole spot;eluent volume,500 μL;eluent,PBS with 1‰ Tween20;elution time,4 hours;elution temperature,room temperature.The results of laboratory evaluation revealed that coincidence rate of the results for testing of HIV/HCV/TP infection were 100%.It had a good analytical specificity;the analytical sensitivity of DBS was a little lower than that of plasma,overally;the C Vs of inter-day,intra-assay and inter-spot of samples of different antibody levels were less than 15%;DBS samples had good stabilities.In clinical evaluation,429 matched plasma/DBS samples were tested.The overall prevalence of HIV,HCV,and TP was 6.1%(26/429),27.7%(119/429)and 15%(64/429).There were 2 HIV/HCV/TP coinfected samples,2 HIV/HCV coinfected samples,4 HIV/TP coinfected samples and 9 HCV/TP coinfected samples.The clinical sensitivity of the assay for anti-HIV,anti-HCV,and anti-TP antibodies was 88.5%(95%CI:0.69～0.97),98.3%(95%CI:0.93～1.00),and 92.2%(95%CI:0.82～0.97),respectively;and the assay was 100%specificity for each analyte.PPV all were 100%;NPV were 99.3%for HIV and it was 98.3%for HCV and 92.2%for TP.The Kappa were 0.96(95%CI:0.86～1.00),0.99(95%CI:0.97～1.00)and 0.95(95%CI:0.91～0.99),respectively.Compared with the routine detection strategy,the results of one-step strategy of DBS as followed:(1)HIV:The coincident rate for positive was 88.5%and negative for 100%;and the total coincident rate was 99.3%(426/429).(2)HCV:The coincident rate for positive was 98.3%and negative for 100%;and the total coincident rate was 99.5%(427/429).(3)TP:The coincident rate for positive was 92.2%and negative for 100%;and the total coincident rate was 98.8%(424/429).2.Establishment and evaluation on the serological method for simultaneously screening for HIV,HCV and TP infection based on one DPSOptimal conditions for DPS sample extraction were:plasma volume,100μL;DPS size,whole spot;eluent volume,500 μL;eluent,PBS with 1‰ Tween20;elution time,2 hours;elution temperature,room temperature.The results of laboratory evaluation revealed that coincidence rate of the results for testing of HIV/HCV/TP infection were100%.It had a good analytical specificity;the analytical sensitivity of DPS was a little lower than that of plasma,overally;the CVs of inter-day,intra-assay and inter-spot of samples of different antibody levels were less than 15%;DPS samples had good stabilities.In clinical evaluation,329 matched plasma/DPS samples were tested.The overall prevalence of HIV,HCV,and TP was 3.0%(10/329)、35.9%(118/329)and 11.2%(37/329),respectively.There were 1 HIV/HCV/TP coinfected samples,2 HIV/HCV coinfected samples,4 HIV/TP coinfected samples and 9 HCV/TP coinfected samples.The clinical sensitivity of the assay for anti-HIV,anti-HCV,and anti-TP antibodies was 90%(95%CI:0.54～0.99),99.2%(95%CI:0.97～1.00),86.5%(95%CI:0.70～0.95),respectively;and the assay was 100%specificity for each analyte.The Kappa were 0.95(95%CI:0.78～1.00),0.99(95%CI:0.98～1.00),0.92(95%CI:0.85～0.99),respectively.Using the plasama results as a reference standard,the results of the comparative analysis of results between DBS and DPS samples as followed:as a whole,the total coincidence rate of DBS and DPS in the detection of HIV/HCV/TP was as follows:(1)in the anti-HIV detection,99.4%(327/329)for DBS and 99.7%(328/329)for DPS;(2)in the anti-HCV detection,99.4%(327/329)for DBS and 99.7%(328/329)for DPS.(3)in the anti-TP detection,98.5%%(324/329)for DBS and 98.8%(325/329)for DPS.Ovrally,the distribution of S/CO value between two types of samples DBS/DPS was similar in HIV,HCV and TP tests and their median value and average value were also approximately equal.3.Establishment,evaluation and application strategy on the molecular method for simultaneously screening for HIV,HCV and TP infection based on one DBSThis study developed the method of screening simultaneously for HIV RNA,HIV DNA and HCV RNA based on one DBS but no for TP DNA.Results showed that:the detection rate of DBS was 71.4%(10/14)when measurable HIV-1 RNA levels were present in plasma(between 1.44～2.99 log10 copies/mL);the detection rate of DBS was 85.7%(6/7)when measurable HIV-1 RNA levels were present in plasma(between 3～3.99 log10 copies/mL);overall,the detection rate of DBS was 84.4%(27/32).The detection rate of DBS was 83.3%(10/12)when measurable HCV RNA levels were present in plasma(between 5～5.99 log10 copies/mL);overall,the detection rate of DBS was 98.1%(101/103).The correlation between HIV-1 VL obtained from plasma and DBS showed that r=0.68(P<0.05);it was that r=0.61(P<0.05)in HCV RNA.Bland-Altman analysis revealed:in HIV-1 RNA,the mean(± SD)difference between HIV-1 RNA in plasma and DBS was 1.00 ± 1.01 logio copies/ml;and all samples were within± 1.96 SDs(-0.97～2.97 log10 copies/mL)for DBS.The mean difference(±SD)in HCV RNA was 0.15 ± 1.08 log10 copies/mL;94.38%(84/89)were within±1.96 SDs(-1.96 to+2.67 log10 copies/mL).On overall,HIV-1 RNA or HCV RNA levels obtained from DBS were always lower than in plasma.HIV-1 DNA in DBS gave concordant results with HIV-1 RNA in plasma.HIV-1 DNA RT-PCR using DBS performed well.Compared with the routine detection strategy,the results of DBS strategy based antibody and nucleic acid tests as followed:the total coincidence rate was 99.3%for HIV-1 RNA and 100%for HIV-1 DNA;98.6%for HCV RNA;98.8%for TP.Conclusion1.The study standardized the protocol of DBS/DPS and establish the ELISA method of screening for anti-HIV,anti-HCV and anti-TP based on one DBS/DPS.This study proved it has a good sensitivity,specificity,and analytical specificity;it has accurate and reliable results.The antibody in DBS/DPS samples was stable.The detection performance of DBS/DPS method were equivalent and they have their own advantages and adapt to different detection environment.It provides reliable and valuable experimental data for the promotion and application of dry blood/plasma spot samples in HIV/HCV/TP detection reagents.2.For the molecular method of screening for HIV-1 RNA/DNA and HCV RNA based one DBS,HIV-1 DNA tests has accurate and reliable detection results.The performance of HIV-1 RNA/DNA needs to be improved.This study proved one DBS can be used for HIV-1 RNA/DNA and HCV RNA tests.3.In this study,a strategy of HIV/HCV/TP antibody and nucleic acid detection combined with DBS was applied to sentinel surveillance of HIV/HCV/TP,which is consistent with the results of the current strategy.It provides a new technical strategy for improving the detection efficiency of sentinel samples,and has an important reference value for establishing a laboratory detection strategy suitable for sentinel surveillance in China. |
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