Objective To evaluate NucliSens HIV-1 QT(bioMerieux,Netherlands) in quantitating human immunodeficiency virus type 1(HIV-1) RNA in whole semen or seminal plasma from HIV-1-infected people.Methods Five levels of HIV-1 RNA were spiked to whole semen,seminal plasma and blood plasma samples from healthy people,and then measured by NucliSens HIV-1 QT.The same method was used to measure HIV-1 RNA in seminal plasma and blood plasma samples from 15 HIV-1 infected people.Results Nucleic acid amplification inhibitors were found in whole semen but not in seminal plasma when using NucliSens HIV-1 QT.No significant difference was found between normal seminal plasma and blood plasma samples spiked with HIV-1 RNA,and no false positive result was found in 10 normal seminal plasma samples.For 15 cases of HIV-1 infection,HIV-1 was detected in 80%(12/15) of the plasma samples and 40%(6/15) of the seminal plasma samples,with viral loads of

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 review and analyze the clinical diagnosis and treatment of renal Ewing's sar-coma with venous tumor embolus,to follow up the survival and prognosis of the patients,and to provide help for the diagnosis and treatment of the disease.Methods:Clinical data(including general data,sur-gical data and postoperative pathological data)of patients diagnosed with renal Ewing's sarcoma with ve-nous tumor embolus in Peking University Third Hospital from June 2016 to June 2022 were collected,and the prognosis of the patients was followed up to analyze the influence of diagnosis and treatment process on the prognosis of the disease.Results:There were 6 patients,including 1 male and 5 females.There were 4 cases of left renal tumor and 2 cases of right renal tumor.The median age at diagnosis was 28 years(16-52 years).The imaging findings were all exogenous tumors with internal necrotic tissue and hemorrhage.The mean maximum tumor diameter was 12.6 cm,and the mean tumor thrombus length was 7.8 cm.Four patients underwent open surgery and 2 patients underwent laparoscopic surgery.The post-operative pathological results were renal Ewing sarcoma.Immunohistochemical results showed 3 cases of CD99(+),2 cases of FLI-1(+),and 1 case of CD99,FLI-1(-).3 patients received chemothera-py(cyclophosphamide,doxorubicin,vincristine/ifosfamide,etoposide),1 case received chemotherapy combined with radiotherapy,and 2 cases received no adjuvant therapy.The mean overall survival(OS)of the 6 patients was 37 months,and the mean OS of the 4 patients(47 months)who received chemo-therapy was significantly higher than that of the 2 patients(16 months)who did not receive chemotherapy(P=0.031).Conclusion:Renal Ewing's sarcoma with venous tumor embolus is rare in clinic,and it is common in young female patients.The operation is difficult and the prognosis is poor.Surgical resection,adjuvant radiotherapy and chemotherapy can improve the overall survival rate of the patients.

Objective An approach for analysis of hepatitis C virus (HCV) quasispecies using Hiseq high-throughput sequencing (hereinafter referred to as Hiseq sequencing) technique was developed and then applied to investigate a possible case of HCV needle sharing transmission. Methods One case of HCV antibody seroconversion (P1) was found in a methadone clinic on January 15, 2015. Four HCV antibody positive injecting drug users (IDUs), P2 to P5, suspected to be involved in needle sharing transmission with P1 during the period (after March 24, 2014) that P1 may be infected with HCV were investigated, and another 28 HCV antibody positive IDUs were selected as controls (C1 to C28). These controls came from the same methadone clinic or lived in the same town with P1. The RNAs were extracted from the plasma specimens and then reverse-transcribed into cDNA. After HCV subtyping, Hiseq sequencing was performed to detect and sequence the HCV quasispecies (263 bp) in the specimens with the same subtype as P1. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated. Results The HCV subtype of specimen P1 was 3b. All the other specimens with the same subtype were P2, C7, C12, C14, C15, C16, C19, C20 and C28. Hiseq sequencing was successfully performed in 9 out of these 10 specimens, and 249 753 to 1 086 333 (average 869 608) cleaned sequences representing 3 to 172 (average 48) unique HCV quasispecies were obtained. The medians (P50) of intrapersonal genetic diversities from the 9 specimens were 0.4% to 12.3%. The P50 (P25, P75) of genetic diversities between P1 and the other 8 specimens were 19.0% (18.4%, 19.8%), 10.4%(2.8%, 18.3%), 19.6% (17.8%, 21.4%),24.9% (23.8%, 26.1%), 19.8% (18.7%, 20.7%), 20.1% (18.9%, 21.2%), 20.6% (20.0%, 21.1%), 23.6% (22.4%, 24.8%). There were no significant difference between the genetic diversities of P1 and P2 and those of P1 and other 7 specimens (H=9.40, P=0.100). The genetic diversities between few HCV quasispecies from P1 and few ones from C7 were 0. Phylogenetic tree analysis indicated that there was no HCV transmission relationship between P1 and P2, but there was HCV transmission relationship between P1 and C7. Conclusion With the feature of high-throughput, easier operation and lower cost, Hiseq sequencing technique has high practical value in tracing HCV transmission at the quasispecies level.

Objective An approach for analysis of hepatitis C virus (HCV) quasispecies using Hiseq high-throughput sequencing (hereinafter referred to as Hiseq sequencing) technique was developed and then applied to investigate a possible case of HCV needle sharing transmission. Methods One case of HCV antibody seroconversion (P1) was found in a methadone clinic on January 15, 2015. Four HCV antibody positive injecting drug users (IDUs), P2 to P5, suspected to be involved in needle sharing transmission with P1 during the period (after March 24, 2014) that P1 may be infected with HCV were investigated, and another 28 HCV antibody positive IDUs were selected as controls (C1 to C28). These controls came from the same methadone clinic or lived in the same town with P1. The RNAs were extracted from the plasma specimens and then reverse-transcribed into cDNA. After HCV subtyping, Hiseq sequencing was performed to detect and sequence the HCV quasispecies (263 bp) in the specimens with the same subtype as P1. The frequency of quasispecies was counted and ranked. Intrapersonal and interpersonal genetic distance and phylogenetic tree were calculated. Results The HCV subtype of specimen P1 was 3b. All the other specimens with the same subtype were P2, C7, C12, C14, C15, C16, C19, C20 and C28. Hiseq sequencing was successfully performed in 9 out of these 10 specimens, and 249 753 to 1 086 333 (average 869 608) cleaned sequences representing 3 to 172 (average 48) unique HCV quasispecies were obtained. The medians (P50) of intrapersonal genetic diversities from the 9 specimens were 0.4% to 12.3%. The P50 (P25, P75) of genetic diversities between P1 and the other 8 specimens were 19.0% (18.4%, 19.8%), 10.4%(2.8%, 18.3%), 19.6% (17.8%, 21.4%),24.9% (23.8%, 26.1%), 19.8% (18.7%, 20.7%), 20.1% (18.9%, 21.2%), 20.6% (20.0%, 21.1%), 23.6% (22.4%, 24.8%). There were no significant difference between the genetic diversities of P1 and P2 and those of P1 and other 7 specimens (H=9.40, P=0.100). The genetic diversities between few HCV quasispecies from P1 and few ones from C7 were 0. Phylogenetic tree analysis indicated that there was no HCV transmission relationship between P1 and P2, but there was HCV transmission relationship between P1 and C7. Conclusion With the feature of high-throughput, easier operation and lower cost, Hiseq sequencing technique has high practical value in tracing HCV transmission at the quasispecies level.