北京市性传播HIV-1感染者流行毒株gag基因序列测定和亚型分析

目的 了解北京市性传播HIV-1感染者流行毒株亚型特点和流行规律.方法 随机采集北京市2008年新确证性传播HIV感染者的抗凝全血标本100份,分离血浆,提取病毒RNA,用套式聚合酶链反应扩增病毒gag基因,并进行序列测定和亚型分析.结果 系统进化分析确定北京市性传播HIV-1感染者流行毒株存在8个亚型或流行重组型,分别为B亚型22份,B'亚型8份,C亚型1份,CRF01_AE 38份,CRF02_AG 2份,CRF07 BC 9份,CRF08_BC 3份,疑似C/CRF01_AE重组型1份.结论 CRF01_AE和B亚型分别占45.2%和26.2%,为性传播感染者主要的亚型,应该加强我市HIV-1亚型流行情况的监测.

Abstract：

Objective To investigate the subtype distribution and sequence characteristics of HIV-1 strains prevalent among sexual infectors in Beijing. Methods We collected the blood samples from 100HIV sexual infectors in Beijing during 2008 and separated plasma specimens. RNA was extracted from the plasma and the gag gene was amplified by RT-PCR and nest-PCR. The PCR products were sequenced directly and phylogenetic analyses of gag gene was performed using the MEGA4 software. Results Among 100 HIV-1 plasma samples,84 gag gene fragments were amplified and analyzed. Eight HIV subtypes including B(22 strains), B'(8 strains),C( 1 strain) ,CRF01_AE (38 strains) ,CRF02_AG (2 strains) ,CRF07_BC(9 strains) ,CRF08_BC(3 strains) and C/CRF01_AE recombinant like strain( 1 strain) were identified circulating in Beijing. Conclusion CRF01 _AE and subtype B were predominant in Beijing account for 45.2% and 26.2% and the surveillance of HIV gene variation should be paid more attention.     

Relationship between the occurrence of virus in plasma and cerebrospinal fluid of HIV-1 infected individuals

Attempts to isolate human immunodeficiency virus type 1 (HIV-1) were carried out on cerebrospinal fluid (CSF) and blood plasma samples from 111 HIV-1 infected subjects in various stages of infection. HIV-1 was recovered at a low rate from CSF of persons with normal immunological parameters but frequently from patients with abnormal values, in all stages of immune system involvement. Isolation from plasma was positive in the majority of the patients, in all stages of infection, with a frequency that was related to the degree of immunodeficiency. HIV-1 could be recovered from the CSF of most patients (74%) with viremia when 85 paired specimens of 58 patients were analyzed. By contrast, HIV-1 was isolated from CSF, but not from plasma, in one case only. HIV-1 p24 antigen measured by an enzyme-linked immunosorbent assay (ELISA) was detectable in only four CSF samples compared with 15 serum samples in paired specimens. These findings indicate that most patients with HIV-1 infection have circulating cell-free infectious virus in the blood and simultaneously demonstrable HIV-1 in the CSF. Replication of HIV-1 exclusively in the central nervous system (CNS) appears to be a rare event.     

Evaluation of the Abbott Real-Time HIV-1 quantitative assay with dried blood spot specimens

The Abbott Real-Time HIV-1 assay was evaluated for its performance in quantification of human immunodeficiency virus type 1 (HIV-1) RNA in dried blood spot (DBS) samples. In total, 169 blood samples with detectable plasma HIV-1 RNA were used to extract RNA from paired DBS and liquid plasma samples, using the automated Abbott m Sample Preparation System (m2000sp). HIV-1 RNA was then quantitated by the m2000rt RealTime analyser. RNA samples suitable for real-time PCR were obtained from all but one (99.4%) of the DBS samples and HIV-1 RNA was detected in 163/168 (97.0%) samples. The correlation between HIV-1 RNA values measured in paired DBS and plasma samples was very high ( r  = 0.882), with 78.5% and 99.4% of cases differing by <0.5 and 1.0 log, respectively. Retesting of DBS replicates following 6 months of storage at 2–8°C showed no loss of HIV-1 RNA in a subset of 89 samples. The feasibility of DBS testing coupled with automated sample processing, and the use of a latest-generation FDA-approved real-time PCR-based system, represents an encouraging first step for viral load measurement in reference centres in developing countries where access to antiretroviral therapy is expanding.     

A simple competitive RT-PCR assay for quantitation of HIV-1 subtype B and non-B RNA in plasma

An easy, inexpensive competitive RT-PCR assay for HIV-1 RNA quantitation was constructed. A 138-bp sequence in the HIV-1 gag p24 region was selected as the target and co-amplified with competitor RNA containing an internal 44-bp deletion. Quantitation of serial dilutions of control RNA samples prepared from the LAI isolate demonstrated a good linearity (R(2)=0.991) within the range between 10 and 250 copies/sample. The detection limit of the assay was determined to be 3.8 copies/sample by Probit analysis and corresponded to 110 copies/ml in plasma. The intra-assay CV value was 9.1%, and the inter-assay value was 25.9%. Both were comparable to those obtained with commercially available HIV-1 RNA quantitation kits. The correlation efficient for the results obtained in 47 plasma samples from HIV-1-infected individuals (subtype A in 1, subtype B in 25, subtype C in 4, subtype F in 1, and CRF01 AE in 16) with the competitive RT-PCR and Cobas Amplicor HIV-1 Monitor test v1.5 was 0.956 for subtype B and 0.947 for subtype non-B. The assay devised is a good alternative for monitoring antiretroviral therapy in resource-poor countries.     

Performance of two commercially available sequence-based HIV-1 genotyping systems for the detection of drug resistance against HIV type 1 group M subtypes

The use of genotyping assays for the detection and evaluation of drug resistance mutations within the polymerase gene of human immunodeficiency virus type 1 (HIV-1) is becoming increasingly relevant in the clinical management of HIV-1 infection. However, genotypic resistance assays available currently have been optimised for genetic subtype B strains of the virus and many clinical centres are presented with strains from subtypes A, C, and D. In the present report, we compare the performance of two sequence-based commercially available kits, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA) and the TruGene HIV-1 Genotyping Kit (Visible Genetics, Toronto, Ontario) against a panel of 35 virus isolates from HIV-1 Group M (subtypes A-J). Full-length consensus sequences were generated by the ViroSeq genotyping system for 26 of 31 (83.8%) of the isolates tested, in contrast to the TruGene genotyping system, which generated 16 of 30 (53%) usable sequences overall. Overall, subtype B isolates were sequenced with a greater degree of success than non-subtype B isolates. Discrepancies were found between the consensus sequences reported by each system for each sample (mean difference 1.0%; range 0.0-3.2%), but these appeared to be random and did not affect interpretation of the major resistance codons. In addition, both systems were able to amplify template RNA from low copy viral load plasma samples (10(2)-10(3) RNA copies/ml) taken from a random selection of patient samples encompassing subtypes A-C. While the availability of these genotyping systems should facilitate studies of HIV-1 drug resistance in countries in which these subtypes are prevalent, the performance against subtypes other than B needs to be improved.     

Improved Single-Copy Assays for Quantification of Persistent HIV-1 Viremia in Patients on Suppressive Antiretroviral Therapy

A quantitative real-time PCR (qRT-PCR) assay with single-copy sensitivity targeting HIV-1 gag RNA (the gag single-copy assay [gSCA]) has been used widely to quantify plasma viremia below the limit of detection of clinical assays in patients on effective antiretroviral therapy (ART), but viral RNA in 15 to 30% of samples amplifies inefficiently because of primer/probe mismatches. We sought to develop improved single-copy assays with increased sensitivity by improving nucleic acid recovery, designing qRT-PCR primers and a probe for a highly conserved region of integrase in the HIV-1 pol gene (the integrase single-copy assay [iSCA]), and increasing the plasma volume tested (Mega-iSCA). We evaluated gSCA versus iSCA in paired plasma samples from 10 consecutive patients with viremia of >1,000 copies/ml and 25 consecutive patients on suppressive ART. Three of 10 viremic samples amplified inefficiently with gSCA compared to the Roche Cobas Ampliprep/TaqMan 2.0, whereas all 10 samples amplified efficiently with iSCA. Among 25 samples from patients on suppressive ART, 8 of 12 samples that were negative for HIV-1 RNA by gSCA had detectable HIV-1 RNA by iSCA, and iSCA detected 3-fold or higher HIV-1 RNA levels compared to gSCA in 10 of 25 samples. Large-volume plasma samples (>20 ml) from 7 patients were assayed using Mega-iSCA, and HIV-1 RNA was quantifiable in 6, including 4 of 5 that were negative by standard-volume iSCA. These improved assays with superior sensitivity will be useful for evaluating whether in vivo interventions can reduce plasma viremia and for assessing relationships between residual viremia and other virologic parameters, including the inducible proviral reservoir.     

BK Virus Characterisation among HIV-1-Infected Individuals and Its Association with Immunosuppression

Purpose: BK virus (BKV) is an opportunistic pathogen which causes significant morbidity and mortality in individuals who are immunodeficient. We aimed to quantitate and characterise BKV and to correlate with the degree of immunosuppression among human immunodeficiency virus (HIV)-1-infected individuals. Methods: BKV DNA detection was carried out using an in-house quantitative real-time polymerase chain reaction on paired whole-blood and urine samples collected from 187 antiretroviral therapy (ART)-naïve HIV-1-infected individuals and 93 healthy individuals who served as controls. Sequencing was performed for a proportion of high BK viral load (VL) samples to observe non-coding control region (NCCR) rearrangements. Results: BKV positivity in urine was 25.6% among HIV-infected individuals and 10.7% in control individuals (P = 0.03). The BK VL showed a significant negative correlation with CD4+ T-cell counts, a positive correlation with WHO clinical staging and no significant correlation with HIV-1 VL. Of 42 BKVs from urine samples sequenced, two showed rearrangements without clinically severe disease or high VL. Their NCCR and VP1 sequence-based genotyping revealed genotype I. In a small subset of individuals (n = 8) on ART who were being followed up, six individuals showed either decrease or complete clearance of virus with ART. Conclusion: There was a higher frequency of BK viruria in HIV-1-infected individuals than among healthy controls and the positivity correlated with the degree of immunosuppression. There was no association of high VL with NCCR rearrangements in urine.     

HIV-1 genetic diversity and transmitted drug resistance among newly diagnosed HIV-1 individuals in Jiangmen,China

Jiangmen is one of the Guangdong-Hong Kong-Macao Greater Bay Areas with frequent commercial intercourse, which is responsible for human immunodeficiency virus type 1 (HIV-1) rapid circulation and genetic evolution for recent years. As a novel HIV-1 second-generation recombinant was previously reported in Jiangmen but the systematic molecular epidemiological investigation was still unknown. A retrospective study on HIV-1 genotypic characteristics and the emergence of transmitted drug resistance in this region was necessary. A total of 224 newly diagnosed HIV-positive cases were randomly selected in Jiangmen City of Guangdong Province between 2018 and 2019. The partial gag (1080 bp), pol (840 bp), and env (460 bp) genes were amplified using nested polymerase chain reaction followed by sequencing. The phylogenetic and recombination analysis as well as HIV-1 drug resistance were performed to surveillance. Sexual transmission was determined to be the major risk factor in Jiangmen. Phylogenetic analysis detected the genotypic distribution as follows: CRF01_AE (36.65%,70 of 191), CRF07_BC (32.46%, 62 of 191), CRF08_BC (4.71%, 9 of 191), CRF55_01B (5.24%, 10 of 191), CRF59_01B (3.14%, 6 of 191), subtype B (4.71%, 9 of 191), subtype C (1.05%, 2 of 191) as well as unique recombinant forms (12.04%, 23 of 191) consisted of seven recombinant patterns, which originated from multiple regions of China. Low-level prevalence of Surveillance Drug Resistance Mutations (2.1%) were predicted but drug-resistant mutations showed at a high level (15.4%) especially mutations in RT gene at position 179 were found to be the most frequent in the therapy-naïve population. Our study highlighted the critical importance of monitoring the emerge of recombinant strains among newly diagnosed HIV-1 individuals along with drug resistance regularly to prevent multi-channel introduction and breakout of new HIV strains.     

Comparison of NucliSens and Amplicor Monitor Assays for Quantification of Human Immunodeficiency Virus Type 1 (HIV-1) RNA in Plasma of Persons with HIV-1 Subtype A Infection in Abidjan, Côte d’Ivoire

We compared the sensitivity and accuracy of the NucliSens assay and those of both the standard and modified (addition of a new primer set, primer mix 1, supplied by Roche) Amplicor HIV Monitor assays to quantify human immunodeficiency virus type 1 (HIV-1) RNA in persons infected with HIV-1 subtype A in Abidjan, Côte d’Ivoire. Seventy-one plasma samples from HIV-1-seropositive persons at different stages of HIV infection and 15 samples from HIV antibody-negative persons were analyzed. The HIV-1 genetic subtype was determined either by DNA sequencing or by a restriction fragment length polymorphism assay. Of the 71 samples, 70 (98%) were subtype A and 1 was subtype G. Of the 70 subtype A samples, the proportion of RNA-positive plasma samples and mean HIV-1 RNA levels were significantly higher by the modified HIV Monitor assay (n = 67 [96%]; mean RNA levels, 5.2 log₁₀ HIV-1 RNA copies/ml) than the NucliSens assay (n = 56 [80%]; 4.3 log₁₀ HIV-1 RNA copies/ml) or the standard HIV Monitor assay (n = 44 [63%]; mean RNA levels, 3.8 log₁₀ HIV-1 RNA copies/ml) (all P values were <0.05). The HIV-1 RNA levels by the modified HIV Monitor assay correlated significantly with those by the NucliSens assay (r = 0.76; P < 0.001) and the standard HIV Monitor assay (r = 0.57; P < 0.001), as did the RNA levels by the NucliSens and the standard HIV Monitor assays (r = 0.60; P < 0.001). Lower CD4 cell counts were significantly correlated with higher HIV-1 RNA levels by all three assays (r = −0.47 for the NucliSens assay, −0.45 for the standard HIV Monitor assay, and −0.62 for the modified HIV Monitor assay). These results indicate that the modified HIV Monitor assay has the highest sensitivity and efficiency at quantifying the levels of RNA in persons infected with HIV-1 subtype A and thus constitutes a valuable tool for the monitoring of RNA levels in areas of Africa were HIV-1 subtype A is predominant.     

A modified ultrasensitive assay to detect quantified HIV-1 RNA of fewer than 50 copies per milliliter

We compared the sensitivity and specificity of versions 1.0 and 1.5 and a modified version 1.5 of the AMPLICOR HIV-1 MONITOR ultrasensitive RNA assay (Roche, Indianapolis, IN) by using a virus stock dilution series and plasma samples from HIV-1-infected and uninfected subjects. The modified assay was linear and consistently positive down to 12 copies per milliliter vs 25 copies per milliliter for the other 2 assays. Versions 1.0, 1.5, and modified 1.5, respectively, detected 9 (23%) of 39, 11 (28%) of 40, and 43 (61%) of 71 replicates of a 4-copy-number and 11 (28%) of 40, 17 (46%) of 37, and 88 (90%) of 98 replicates of a 10-copy-number standard. Of 44 patient samples with undetectable levels using version 1.0, 32 (73%) had detectable levels on the modified assay, and 5 (25%) of 20 had detectable levels on version 1.5. None of the assays detected HIV-1 RNA in HIV-1 antibody-negative samples. The modified version 1.5 of the RNA assay is more sensitive for detecting HIV-1 RNA in significantly more patients than are versions 1.0 and 1.5.     

Quantification of genital human immunodeficiency virus type 1 (HIV-1) DNA in specimens from women with low plasma HIV-1 RNA levels typical of HIV-1 nontransmitters

Studies of human immunodeficiency virus type 1 (HIV-1) transmission suggest that genital HIV-1 RNA and DNA may both be determinants of HIV-1 infectivity. Despite its potential role in HIV-1 transmission, there are limited quantitative data on genital HIV-1 DNA. Here we validated an in-house real-time PCR method for quantification of HIV-1 DNA in genital specimens. In reactions with 100 genomes to 1 genome isolated from a cell line containing one HIV-1 provirus/cell, this real-time PCR assay is linear and agrees closely with a commercially available real-time PCR assay specific for a cellular housekeeping gene. In mock genital samples spiked with low numbers of HIV-1-infected cells such that the expected HIV-1 DNA copy number/reaction was 100, 10, or 5, the average copy number/reaction was 80.2 (standard deviation [SD], 28.3), 9.1 (SD, 5.4), or 3.1 (SD, 2.1), respectively. We used this method to examine genital HIV-1 DNA levels in specimens from women whose low plasma HIV-1 RNA levels are typical of HIV-1 nontransmitters. The median HIV-1 DNA copy number in endocervical secretions from these women (1.8 HIV-1 DNA copies/10,000 cells) was lower than that for women with higher plasma HIV-1 RNA levels (16.6 HIV-1 DNA copies/10,000 cells) (P=0.04), as was the median HIV-1 DNA copy number in vaginal secretions (undetectable versus 1.0 HIV-1 DNA copies/10,000 cells). These data suggest that women with low plasma HIV-1 RNA and thus a predicted low risk of HIV-1 transmission have low levels of genital HIV-1 cell-associated virus. The assay described here can be utilized in future efforts to examine the role of cell-associated HIV-1 in transmission.     

Quantitation of HIV-1 RNA levels in plasma and CSF of asymptomatic HIV-1 infected patients from South India using a TaqMan real time PCR assay.

BACKGROUND: Most of the quantitation assays for HIV-1 RNA used currently are designed and optimized for HIV-1 subtype B viruses and hence may not be suitable for India, where the predominant subtype is HIV-1 subtype C. OBJECTIVES: Development and standardization of HIV-1 TaqMan real time PCR assay suitable for measuring plasma and CSF viral RNA levels in HIV subtype C infected individuals. STUDY DESIGN: A TaqMan real time PCR was developed using primers and probes selected in the gag region for detection of Indian HIV-1 subtype C strain. Plasma (n=120) and CSF samples (n=46) obtained from HIV infected subjects were used to evaluate the sensitivity and specificity of the assay. A comparative evaluation was carried out with a commercially available quantitative HIV viral load assay (Roche Amplicor Version 1.5). RESULTS: The TaqMan assay was able to amplify all HIV-1 group M subtypes except subtype E. Viral loads could be estimated in all the plasma (n=120) and 40/46 CSF samples obtained from HIV positive subjects. Sensitivity of this assay was found to be 180 copies/ml. Correlation with the commercially available viral load assay was very good (r=0.885). CONCLUSIONS: A TaqMan real time PCR was standardized for HIV-1 subtype C and it was more sensitive (180 copies/ml) than standard Amplicor monitor assay, Version 1.5 (400 copies/ml).     

Human Immunodeficiency Virus Type 1 Subtypes Prevalence in Central China

Purpose

To study the epidemic characteristics, transmission sources and routes of various subtypes of human immunodeficiency virus type 1 (HIV-1) and sequence variations in Henan, central China. To provide theoretical foundation for Acquired Immune Deficiency Syndrome (AIDS) prevention strategy in this region where the primary HIV transmission route was through former paid blood donation.

Materials and Methods

HIV-1 gene env and gag were amplified by nested polymerase chain reaction (PCR) from uncultured peripheral blood mononuclear cells (PBMCs) obtained from 1,287 HIV-1 confirmed samples in Henan.

Results

Among 1,287 samples, 5 HIV-1 strains were found including subtypes B'' (95.9%), C (0.47%) and recombinant subtypes CRF 07_BC (1.09%), CRF 08_BC (1.79%) and CRF 01_AE (0.78%). Phylogenetic tree analysis found that 1,234 Henan subtype B'' were closely related to those commonly found in Thailand, and were distantly related to other international subtypes. The dominant strain in former blood plasma donors (FPDs) was subtype B'', and the dominant strains in sexual transmission were subtype B'' and BC. Among HIV patients who were most likely infected through routes other than paid blood donation, the percentage of non-B'' subtypes was much higher than those of FPD.

Conclusion

These findings suggest that the prevailing strain of HIV-1 in Henan is subtype B'', similar to the B'' subtype found in Thailand. In addition, for the first time we found subtypes C and recombinant subtypes CRF07_BC, CRF08_BC and CRF01_AE in this region. Indicating that the subtype feature of HIV-1 became more complicated than before in central China.     

Evaluation of NucliSens EasyQ HIV-1 assay for quantification of HIV-1 subtypes prevalent in South-east Asia.

BACKGROUND: Monitoring anti-retroviral therapy requires that viral load assays for human immunodeficiency virus type 1 (HIV-1) be applicable to diverse HIV-1 subtypes. OBJECTIVES: To evaluate NucliSens EasyQ HIV-1 assay for quantitation of common HIV-1 subtypes prevalent in South-east Asia. STUDY DESIGN: One hundred and nineteen plasma samples collected in Hong Kong and Cambodia were used to compare the performance of NucliSens EasyQ HIV-1 and COBAS Amplicor HIV-1 Monitor version 1.5 assays. Viral RNA extracted from the NucliSens MiniMAG was also used for HIV-1 subtyping. RESULTS: Performance of NucliSens EasyQ correlated well with COBAS Amplicor (r=0.777, p<0.001) and the small mean difference (0.0462log(10)IU/mL) obtained in the Bland and Altman model indicated good agreement between two assays. The NucliSens EasyQ assay demonstrated a 95% sensitivity at 500IU/mL and 100% specificity. Reproducibility of this assay was within log(10)2-4IU/mL and had a coefficient of variation between 2.3% and 10.4%. Among the 109 specimens included in the analysis, HIV-1 subtyping identified 64 CRF01_AE, 38 subtype B, 3 subtype C, 3 CRF07_BC and 1 subtype G viruses. CONCLUSIONS: Performance of NucliSens EasyQ was comparable to COBAS Amplicor for HIV-1 viral load monitoring. RNA extracts from NucliSens MiniMAG could be used for HIV-1 viral load monitoring, subtyping and drug resistance mutations detection. Our findings highlight the versatility of both NucliSens EasyQ and COBAS Amplicor in monitoring prevalent subtypes and rare circulating recombinant forms (CRFs) in the South-east Asia region.     

Performance of the Abbott RealTime HIV-1 assay for quantification of HIV-1 clades prevalent in China.

OBJECTIVE: To evaluate the performance of the Abbott RealTime HIV-1 assay for measuring viral loads of the prevalent HIV-1 clades in China. STUDY DESIGN: Serially diluted samples, as well as 521 clinical samples from 213 untreated HIV-1-infected individuals, 56 HIV-1-infected patients receiving ART, 60 HCV- and 57 HBV-infected patients and 135 healthy blood donors, were tested with RealTime and EasyQ. RESULTS: Both assays exhibited linearity coefficients of >0.98. RealTime and EasyQt detected HIV-1 RNA in 87.36% and 86.99% of 269 HIV-1-seropositive samples, respectively. The correlation coefficients between the two assays for quantifying HIV-1 clades B', BC and AE were 0.884, 0.813 and 0.881, respectively, and the mean differences between the two assays for clades B', BC and AE were -0.087, 0.314 and 0.559 log(10)IU/mL, respectively. The correlation coefficient between the two assays for measuring samples from patients receiving ART was 0.945 (mean difference, 0.085 log(10)IU/mL). No false-positive samples were found among the 60 HCV-infected patients, 57 HBV-infected patients and 135 healthy blood donors. CONCLUSIONS: The Abbott RealTime HIV-1 assay shows good linearity and specificity. ART drugs and HIV-1 clades B' and BC do not affect the performance of the assay. Based on the comparison data, [corrected] clade AE may be more [corrected] readily detected by using this method [corrected]