Objective To evaluate the application of pooling HIV nucleic acid amplification testing (NAAT) among men who had sex with men (MSM) population, and to investigate suitable HIV screening strategy and the feasibility of calculation of HIV incidence using pooling NAAT among MSM population in China.Methods Four thousand eight hundred and fifty-six samples were collected from MSM population from April 2008 to September 2009 among with 4 156 were in Heilongjiang province and 700 were in Beijing in China. After standard testing with an HIV ELISA and WB confirmation testing, HIV antibody-negative samples were pooled and screened for HIV using NAAT.A three-stage pooling strategy was adopted.The HIV positive rate estimated by the four HIV screening strategies was calculated.In addition, 4 156 HIV positive specimens from Heilongjiang province were screened with the BED capture enzyme immunoassay (BED-CEIA).The HIV-1 incidences were estimated by BED-CEIA assay and pooling NAAT individually.ResultsOne hundred and forty-three of 4 856 subjects were HIV infected.130 were 3rd and 4th generation ELISA positive; 13 were antibody-negative but acutely HIV infected.According to the evaluation of four HIV screening strategies, routine HIV screening test together with pooling NAAT was more effective than other strategies for screening out window period generation ELISA+WB+pooling NAAT' were 2.68%(95% confidence interval CI=2.22%-3.14%), 2.82%(95%CI=2.35%-3.29%), 2.94%(95%CI=2.46%-3.42%) and 2.94%(95%CI=2.46%-3.42%), respectively.The differences were not significant (χ2=0.854 3, P=0.836 4).Of the 88 HIV positive samples from Heilongjiang province, 44 participants were tested as recent HIV infections by BED-CEIA assay. The estimated HIV-1 incidence was 2.36% (95%CI=1.63%-3.08%) and 2.92% (95%CI=1.01%-4.83%) based on BED-CEIA assay and pooling NAAT,respectively.Conclusions Pooling NAAT is a effective screening test in HIV negative population to detect window period infection among MSM population in China.

4. 0 Log was precede 5 DBS samples whose plasma VL

ObjectiveTo establish a novel assay for HIV-1 p24 ultrasensitive detection based on Gold Nanoparticle Probe (GNP) and PCR.MethodsSandwich ELISA method was established by a pair of anti-p24 monoclonal antibodies (mAbs),1G12 and 1D4,and was used to detect recombinant HIV-1 p24 antigen.The bio-barcode DNA was 47 bp,selected from genome of Arabidopsis,and formed double-stranded DNA by hybridization with the capture DNA (complementary with bio-barcode DNA) modified with sulfhydryl.Then double-stranded DNA were conjugated on the surface of 1D4-modified gold nanoparticles by sulfhydryl,and the Gold Nanoparticle Probe was produced.1G12 was precoated in the micropaltes,and in the presence of target recombinant HIV-1 p24 protein,a sandwich immuno-complex would form by adding GNP.Then the bio-barcode DNA in the immuno-complex were released by heating as detection signal,and consequently characterized by the polymerase chain reaction (PCR) with synthesized special primers and analyzed by 4％ agar gel electrophoresis,so HIV-1 p24 antigen could be evaluated.The sensitivity comparison between the new assay and ELISA can be done.ResultsSandwich ELISA was used to quantify HIV-1 p24 antigen by monoclonal antibodies 1G12 and 1D4,and the limit of detection (LOD) was 1000 pg/ml.The new GNP assay was established by the same pair of antibodies,combined with PCR and agar gel electrophoresis,and was used to indirectly detect HIV-1 p24 antigen.The band intensity of PCR products paralleled with the quantity of HIV-1 p24 antigen,and the limit of detection (LOD) could reach down to 1 pg/ml.ConclusionThe new assay based on GNP and PCR was efficient in the detection of HIV-1 p24,which is at least 3 orders of magnitude more sensitive than traditional ELISA.

OBJECTIVEAn approach for analysis of HIV quasispecies using Miseq high-throughput sequencing platform (hereinafter referred to as Miseq platform) was established and applied to contact tracing for a possible case of HIV sexual transmission.

METHODSFour plasma specimens were collected from 2 HIV infections (P1 and P2) suspected to be involved in the sexual transmission and 2 local HIV infections as controls (P3 and P4). The RNAs were extracted from the specimens and then reverse-transcribed into cDNA. After HIV subtyping, Miseq platform was performed to detect and sequence the HIV quasispecies (352 bp) in each specimen. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated by using the top 5, 20, 100, 500, and all quasispecies, respectively.

RESULTSThe subtypes of HIV from all 4 specimens were CRF01_AE. 23 788 to 37 397 cleaned sequences representing 1 229 to 1 412 unique HIV quasispecies were obtained from these specimens by using Miseq platform. The average genetic distance (3.5%-4.5%) between quasispecies from specimens P2 and P1 was significantly lower than that (10.3%-19.6%) between quasispecies from P2 and the controls (P3 or P4). Phylogenetic tree analysis indicated that sequences from specimens P1 and P2 clustered together while sequences from P3 and P4 exhibited completely independent clusters. When the top 20 or more quasispecies from each specimen were analyzed, sequences from P1 showed a paraphyletic relationship with those from P2, which may indicated that the direction of HIV transmission was from P1 to P2.

CONCLUSIONWith the feature of convenient and economic operation, Miseq platform has high practical value in contact tracing of possible HIV transmission.

Contact Tracing ; HIV Infections ; HIV Seropositivity ; HIV-1 ; Humans ; Phylogeny

Objective To compare the bio-equivalence among commercial HIV-1 viral load tests,including EasyQ HIV-1 v2.0 (EasyQ) from bioMerieux NucliSens of France;VERSANT HIV-1 RNA 3.0 assay (bDNA) from Siemens Healthcare Diagnostics of USA;COBAS AmpliPrep/COBAS TaqMan HIV-1 test (Taqman) from Roche Molecular Diagnosis of USA;Abbott Real Time HIV-1 Kit (M2000) from Abbott Molecular of USA and two domestic HIV-1 viral load test kits (domestic kit) from DaAn Gene Company of Sun Yat-Sen University and Liaoning Bio-Pharmaceutical company of Northeast pharmaceutical group,by using proficiency test results in China from 2013 to 2015.Methods A total of 2 954 proficiency test results,obtained from 22 positive samples of 6 proficiency tests in 155 laboratories conducted by China CDC were analyzed during 2013-2015.The results from each sample were first logarithmic transformed and then grouped according to the method used,the mean value of logarithmic results was calculated.Subsequently,22 clusters of mean values were analyzed by Bland-Altman analysis for the consistency,and linear regression analysis for the interdependency.Results The results indicated that,by taking Taqman as the reference,EasyQ,M2000,bDNA and domestic kit had good consistency (90％-100％) and interdependency.Conclusion All the viral load tests were bio-equivalent.Moreover,according to the conversion formula derived from domestic proficiency test results,all the viral load results could be converted,which is critical for epidemiological analysis.

Objective To compare the community viral load (CVL) among MSM in 15 cities in China using standardized national reference sources.Methods The study analyzed the existing database of National Major Science and Technology Project of China.The database was established with serial random survey of MSM HIV CVL among MSM in 15 cities from 2013 to 2015.VL tests were conducted in 15 laboratories with different equipment and methods,including RT-PCR,nucleic acid sequence based amplification (NASBA),branched DNA testing (bDNA) and Abbott M2000 RealTime system (M2000).Based on proficiency test for 15 laboratories conducted by National HIV Reference Laboratory,VL test values detected with EasyQ,bDNA and M2000 were converted and standardized into resultant values of TaqMan 2.0.Software SPSS 17.0 was used to produce descriptive statistics for the dataset.Results From 2014 to 2015,the 15 testing sites were found to use a number of different viral load detection techniques.In 2014,the community viral load values were (2.38 ±1.47) and (2.99 ± 1.31) in 15 testing sites,while in 2015 these values were found to be (2.07± 1.34) and (2.72± 1.19).The measurement of community VL was done using standard benchmarks of ≤200 copies/ml,≤400 copies/ml and ≤1 000 copies/ml,that were used for reference for now.Conclusion It is necessary to use standard detection method to improve the comparability of annual results.Using a standardized rate of ≤400 copies/ml or ≤ 1 000 copies/ml for successful control of VL was found with high stability for the result comparison among different areas.