Rapid and automated tetrazolium-based colorimetric assay for the detection of anti-HIV compounds

A rapid, sensitive and automated assay procedure was developed for the in vitro evaluation of anti-HIV agents. An HTLV-I transformed T4-cell line, MT-4, which was previously shown by Koyanagi et al. (1985) to be highly susceptible to, and permissive for, HIV infection, served as the target cell line. Inhibition of the HIV-induced cytopathic effect was used as the end point. The viability of both HIV-and mock-infected cells was assessed spectrophotometrically via the in situ reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The procedure was optimized as to make optimal use of multichannel pipettes, microprocessor-controlled dispensing and optical density reading. The absorbance ratio of the mock-infected control to the HIV-infected samples was about 20. This allowed an accurate determination of the 50% effective doses, as demonstrated for 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-dideoxycytidine (ddCyd), dextran sulfate and heparin. The technique significantly reduced labor time as compared to the trypan blue exclusion method, and permits the evaluation of large numbers of compounds for their anti-HIV activity.     

Multicenter evaluation of Xpert HIV-1 viral load assay for HIV quantification in China

New approaches to increase HIV-1 testing and HIV-1 viral load (VL) monitoring are needed for people living with HIV (PLHIV) in China. The Xpert HIV-1 VL assay was prequalified by the World Health Organization in 2017 but has not been evaluated in China. A multicenter evaluation was conducted to assess the accuracy of the Cepheid Xpert HIV-1 VL assay compared to the Abbott RealTime HIV-1 assay in China. Overall agreement was seen in 558 of 562 specimens (99.29%) with a κ value of 0.962. Pearson's coefficient between the two assays was 0.943. Analyzed by the Bland-Altman method, the mean bias was −0.54 log₁₀ copies/mL, and 94.05% results fell within the 95% confidence limit of agreement (−1.248 to 0.168 log₁₀ copies/mL). The coefficient of variation of the Cepheid Xpert HIV-1 VL assay ranged from 0.61% to 1.55%, as determined by testing eight positive plasma specimens with three different lots on different days. Due to its simplicity, random-access, rapid turnaround time, and accuracy, the Xpert HIV-1 VL assay can be used in local hospitals and clinics that bear the burden of identifying and treating HIV patients in China.     

Detection and quantification of HIV-1 RNA with a fully automated transcription-mediated-amplification assay

BackgroundNucleic acid testing is the major method used to monitor HIV viral load. Commercial systems based on real-time PCR assays are available for high-volume centralized laboratory testing, but they are not fully automated.Objectives and study designWe have compared the diagnostic performance of the Hologic Aptima HIV-1 Quant Dx assay (Aptima) (based on real-time TMA) on the Panther instrument, a fully-automated random access platform, to that of, the Roche Cobas Ampliprep Cobas TaqMan (CAP/CTM) HIV-1 version 2.0 (based on real-time PCR).ResultsProbit analysis of replicate dilutions of NIBSC WHO International HIV-1 Standard, gave LODs of 8.6 c/ml for Aptima and 15.2 c/ml for CAP/CTM. The agreement between the assays was excellent when measuring HIV RNA in a calibrated reference (κ = 0.90, p < 0.001) and good when measuring clinical samples (κ = 0.62, p < 0.001). The correlation among the samples quantified by the two methods was very good (r = 0.95, p< 0.001) and the mean difference between the values obtained with the two assays was 0.02 log c/ml for B and non-B subtypes. The vast majority of results showed <0.5 log variance between the two assays (89%); only one sample showed results that differed by over 1.0 log c/ml.ConclusionThe performance of the new fully automated Aptima assay is adequate for clinical monitoring of HIV-1 RNA during infections and treatment. The Aptima assay is well suited for routine laboratory use.     

Seroepidemiology of WU polyomavirus among children exposed perinatally to HIV-1

WU polyomavirus (WUPyV), a new member of the genus wukipolyomavirus in the family Polyomaviridae, has been detected in serum and tissues of individuals infected with HIV. However, the epidemiology of WUPyV among children exposed perinatally to HIV‐1 is unknown. To investigate the epidemiology of WUPyV in children exposed to and infected perinatally with HIV, serum samples from 150 children exposed to HIV and 114 children infected with HIV were screened for IgG antibodies to WUPyV. A subset was screened for IgM antibodies to WUPyV. Both antibody detection assays were performed using a recombinant WUPyV VP1‐based ELISA. The overall seroprevalence of WUPyV IgG was 76.3% in children infected with HIV and 62% in children exposed perinatally to HIV. In the group of children infected with HIV, the prevalence of WUPyV IgG antibody reached its peak in 2–3 year olds (90.9%). In children 0–5 months of age, IgG seroprevalence was lower in those children exposed to HIV compared to children infected with HIV (43.1% vs.75%, P = 0.047). However, the seroprevalence of WUPyV IgM antibody was higher in children exposed to HIV compared to infants infected with HIV (27.4% vs. 8.3%, P = 0.044). WUPyV infection is acquired in early childhood in the majority of children born to mothers infected with HIV. The implication of this infection and the specific clinical syndrome that it produces, if any, remain to be defined. J. Med. Virol. 84:188–193, 2012. © 2011 Wiley Periodicals, Inc.     

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]     

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.     

A highly sensitive and specific time resolved fluorometric bridge assay for antibodies to HIV-1 and -2

This study addresses the continuing need to develop human immunodeficiency virus-1 (HIV-1) and HIV-2 immunoassays with increased sensitivity. Two chimeric antigens, r-HIV-1env, incorporating immunoreactive regions of HIV-1 glycoprotein (gp) 120 and gp41, and r-HIV-2env, incorporating HIV-2 gp125 and gp36, and their corresponding in vivo biotinylated versions, r-Bio-HIV-1env and r-Bio-HIV-2env, were expressed in Escherichia coli and purified by single step affinity chromatography. These antigens were used to set up a bridge assay for the detection of anti-HIV antibodies. Anti-HIV-1 and HIV-2 antibodies in sera were captured using a mixture of the biotinylated antigens, immobilized on streptavidin-coated microtiter wells, and revealed using a mixture of the non-biotinylated antigens, labeled with either Eu³⁺ chelate or with nanoparticles doped with the Eu³⁺ chelate, followed by fluorescence measurement using time resolved fluorometry (TRF). The performance of this TRF immunoassay was compared to that of five commercial HIV ELISAs using well-characterized sera panels. The results show that the TRF immunoassay using either form of the label was in complete agreement with the commercial assays. The use of the Eu³⁺ chelate label enhanced sensitivity significantly when used in the nanoparticle format as evidenced by the very high signal-to-cut-off ratios.     

Quantification of HIV-1 proviral DNA by a standardized colorimetric PCR-based assay

A simple method was developed for measuring human immunodeficiency virus type 1 (HIV-1) proviral DNA in mononuclear cells based on the commercially available Amplicor(TM) HIV-1 polymerase chain reaction (PCR) assay and the limiting dilution method. The lowest limit of detection was four proviral genomes per 10⁶ cells. The accuracy was demonstrated by using serial dilutions of LAV-8E5 cells, and the interassay variability was 0.2 log. The technique was used to measure HIV-1 proviral DNA in the peripheral blood mononuclear cells (PBMC) of 18 antiretroviral drug-naive HIV-1-positive individuals before and 4 weeks after initiating double nucleoside therapy. The DNA proviral titers at baseline (median = 3.45, range = 2.11–4.7 log copies/10⁶ cells) were 2.08 log greater than the infectivity titers, but there was a correlation between these two parameters (r = 0.63, P = 0.009). The mean decrease in the proviral DNA titer after 4 weeks of therapy was 0.31 log, whereas the decrease in the infectivity titer was 0.81 log and the decrease in the plasma RNA concentration was 1.29 log. The technique was also used to measure HIV-1 proviral DNA in the PBMC of 11 patients who had undetectable plasma HIV-1 RNA after being placed on combination antiretroviral therapy. Although proviral DNA remained detectable in all patients after 36 weeks of treatment, a gradual decline with an estimated half-life of 21–58 weeks was observed. The reliability of this simple and convenient colorimetric PCR-based technique indicates its suitability for assessing the effect of current antiretroviral regimens on the latent reservoirs of provirus. J. Med. Virol. 54:54–59, 1998. © 1998 Wiley-Liss, Inc.     

Significance of plasma and peripheral blood mononuclear cell derived HIV-1 sequences in establishing epidemiologic linkage between two individuals multiply exposed to HIV-1

Establishing epidemiologic linkage in individuals multiply exposed to HIV can be a difficult task. To date, only peripheral blood mononuclear cell (PBMC)-derived sequences have been used in studying HIV-1 transmission between individuals. So far, the combined utility of plasma and PBMC-derived HIV-1 sequences has not been assessed in establishing epidemiologic linkage in people involved in transmission of HIV. In this study, both PBMC (DNA) and plasma (RNA) derived viral quasispecies was used in establishing epidemiologic linkage between two infected individuals (B-90 and B-69) multiply exposed to HIV-1 via injecting drug use. A detailed sequence, and phylogenetic analyses of HIV-1V3 region quasispecies derived from these two compartments clearly demonstrated compartmentalization of viral quasispecies between PBMC and plasma. More importantly, these data also demonstrate that in order to establish epidemiologic linkage between individuals multiply exposed to HIV-1, analyses of viral strains from both plasma and PBMC compartments may be necessary. The PBMC compartment alone may not provide sufficient information on epidemiologic linkage, overall diversification of viral quasispecies, replacement of older strains and the emergence of new viral recombinant strains in vivo. These are the first analyses that demonstrate the incremental value of plasma derived sequences, when used in conjunction with PBMC-derived sequences, in establishing the epidemiologic linkage between individuals multiply exposed to HIV parenterally. Further, the plasma derived HIV-1 sequences may prove to be invaluable in predicting a recent transmission between two epidemiologically-linked individuals.     

A rapid colorimetric assay to evaluate the effects ofBacillus thuringiensis toxins on cultured insect cells

Summary A new cytotoxicity assay has been developed to assess the activity of the toxins produced byBacillus thuringiensis kurstaki on CFl cells. The assay is performed in a 96-well plate using a tetrazolium salt, MTT, as the substrate. The compound diffuses through the cell membrane and is reduced by mitochondrial dehydrogenases of live cells to a purple color compound, which precipitates within the cells. The precipitate is dissolved by isopropanol and the absorbance at 570 nm is measured with a 96-well plate reader. The amount of dye converted is proportional to the number of liver cells and the length of incubation with substrate. The LC₅₀ of the 60 kDa toxin, an activated form of the 130 kDaB. t. kurstaki toxin has been determined and found to be comparable to the data obtained by the trypan blue staining of dead cells.     

HIV-1 integrates into resting CD4+ T cells even at low inoculums as demonstrated with an improved assay for HIV-1 integration

Human Immunodeficiency Virus Type 1 (HIV-1) establishes a latent reservoir early in infection that is resistant to the host immune response and treatment with highly active antiretroviral therapy (HAART). The best understood of these reservoirs forms in resting CD4(+) T cells. While it remains unclear how reservoirs form, a popular model holds that the virus can only integrate in activated CD4(+) T cells. Contrary to this model, our previous results suggest that HIV-1 can integrate directly into the genomes of resting CD4(+) T cells. However, a limitation of our previous studies was that they were conducted at high viral inoculum and these conditions may lead to cellular activation or saturation of restriction factors. In the present study, we tested if our previous findings were an artifact of high inoculum. To do this, we enhanced the sensitivity of our integration assay by incorporating a repetitive sampling technique that allowed us to capture rare integration events that occur near an Alu repeat. The new technique represents a significant advance as it enabled us to measure integration accurately down to 1 provirus/well in 15,000 genomes--a 40-fold enhancement over our prior assay. Using this assay, we demonstrate that HIV can integrate into resting CD4(+) T cells in vitro even at low viral inoculum. These findings suggest there is no threshold number of virions required for HIV to integrate into resting CD4(+) T cells.     

The Multispot rapid HIV-1/HIV-2 differentiation assay is comparable with the Western blot and an immunofluorescence assay at confirming HIV infection in a prospective study in three regions of the United States

BackgroundA new HIV diagnostic algorithm has been proposed which replaces the use of the HIV-1 Western blot and HIV-1 immunofluorescence assays (IFA) as the supplemental test with an HIV-1/HIV-2 antibody differentiation assay.ObjectivesTo compare an FDA-approved HIV-1/HIV-2 antibody differentiation test (Multispot) as a confirmatory test with the HIV-1 Western blot and IFA.Study designParticipants were screened with an HIV-1/HIV-2 combination Antigen/Antibody (Ag/Ab) screening assay. Specimens with repeatedly reactive results were tested with Multispot and either Western blot or IFA. Specimens with discordant screening and confirmatory results were resolved with HIV-1 RNA testing.ResultsIndividuals (37,876) were screened for HIV infection and 654 (1.7%) had a repeatedly reactive Ag/Ab assay result. On Multispot, 554 (84.7%) were HIV-1 reactive, 0 (0%) were HIV-2 reactive, 1 (0.2%) was reactive for both HIV-1 and HIV-2 (undifferentiated), 9 (1.4%) were HIV-1 indeterminate, and 90 (13.8%) were non-reactive. HIV-1 RNA was detected in 47/90 Multispot non-reactive (52.2%) specimens. Among specimens confirmed to have HIV infection (true positives), Multispot and Western blot detected HIV-1 antibody in a similar proportion of cases (93.7% vs. 94.4% respectively) while Multispot and IFA also detected HIV-1 antibody in a similar proportion of cases (84.5% vs. 83.4% respectively).ConclusionsIn this study, Multispot confirmed HIV infections at a similar proportion to Western blot and IFA. Multispot, Western blot, and IFA, however, did not confirm all of the reactive Ag/Ab assay results and underscores the importance of HIV NAT testing to resolve discordant screening and confirmatory results.     

实时荧光环介导等温扩增检测 HIV-1 DNA反应体系的建立

目的建立一种快速、敏感度高的实时荧光环介导等温定量检测HIV-1 DNA的方法。方法根据HIV病毒基因序列选择6个保守区域设计4条环介导等温扩增（ LAMP）引物,建立环介导等温扩增体系,同时在体系中加入SYBR GreenⅠ荧光染料,并评价该方法的特异性和敏感度。结果成功合成实时荧光环介导等温定量检测HIV-1 DNA反应体系,检测200例HIV感染者外周淋巴细胞HIV-1 DNA,其中有195例阳性,并检测50例健康对照结果均阴性。其中有HIV-1 DNA定量结果显示最低含量为51拷贝/ml,最高含量为8．21×106拷贝/ml,平均值含量为5．78×105拷贝/ml,其中病毒含量比较集中在105数量级范围,共162例占83．08％。采用10倍稀释法发现该方法最低检出限为50拷贝/ml。对乙型肝炎病毒、疱疹病毒、丙型肝炎病毒进行交叉实验结果均为阴性。结论HIV感染者外周血中几乎都存在HIV-DNA,实时荧光环介导等温扩增是一种快速、敏感度高、特异性强的方法,适合各级医院的临床检测。     

A global approach to HIV-1 vaccine development

A global human immunodeficiency virus-1 (HIV-1) vaccine will have to elicit immune responses capable of providing protection against a tremendous diversity of HIV-1 variants. In this review, we first describe the current state of the HIV-1 vaccine field, outlining the immune responses that are desired in a global HIV-1 vaccine. In particular, we emphasize the likely importance of Env-specific neutralizing and non-neutralizing antibodies for protection against HIV-1 acquisition and the likely importance of effector Gag-specific T lymphocytes for virologic control. We then highlight four strategies for developing a global HIV-1 vaccine. The first approach is to design specific vaccines for each geographic region that include antigens tailor-made to match local circulating HIV-1 strains. The second approach is to design a vaccine that will elicit Env-specific antibodies capable of broadly neutralizing all HIV-1 subtypes. The third approach is to design a vaccine that will elicit cellular immune responses that are focused on highly conserved HIV-1 sequences. The fourth approach is to design a vaccine to elicit highly diverse HIV-1-specific responses. Finally, we emphasize the importance of conducting clinical efficacy trials as the only way to determine which strategies will provide optimal protection against HIV-1 in humans.     

Evolution of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy from 2000 to 2010

Prevalence and predictors of transmitted drug resistance (TDR), defined as the presence of at least one WHO surveillance drug resistance mutation (SDRM), were investigated in antiretroviralna?ve HIV-1-infected patients, with a genotypic resistance test (GRT) performed ≤6 months before starting cART between 2000 and 2010. 3163 HIV-1 sequences were selected (69% subtype B). Overall, the prevalence of TDR was 12% (13.2% subtype B, 9% non-B). TDR significantly declined overall and for the single drug classes. Older age independently predicted increased odds of TDR, whereas a more recent GRT, a higher HIV-RNA and C vs. B subtype predicted lower odds of TDR.     

A novel assay to identify entry inhibitors that block binding of HIV-1 gp120 to CCR5

HIV-1 infection is initiated by the interaction of the envelope glycoprotein gp120 with the cellular receptor CD4 that triggers conformational changes in gp120 necessary for subsequent interaction with a coreceptor CCR5 (or CXCR4). The CD4-induced (CD4i) conformation of gp120 can be mimicked by a full-length single chain (FLSC) protein consisting of gp120 linked with the D1D2 domains of CD4 by a 20-amino-acid linker. We have used this protein to establish a flow cytometry-based assay and an ELISA-based assay to identify inhibitors that block the binding of gp120 to CCR5. Both assays are specific for detecting the known CCR5 antagonist TAK-779, but the ELISA-based assay was more sensitive, simple, inexpensive, and rapid; thus, it can be adapted to high throughput screening (HTS). The ELISA-based method was validated with a diverse set of known antagonists, for example, TAK-779, AOP-RANTES, PSC-RANTES, and several mAbs.     

A rapid, automated colorimetric assay for measuring antibody binding to cell surface antigens

An automated, colorimetric procedure is described for detecting antibodies specific for cell surface antigens. The procedure entails (a) coating the wells of 96-well microplates with either protein A or anti-immunoglobulin antibodies and (b) preincubating either the microplate or target cells with the test antibody. Target cells which react with the test antibody bind to the wells of the microplate and bound cells are quantitated by staining with the dye Rose Bengal. A microplate spectrophotometer is used to measure absorbance in each well of the plate, providing a rapid, automated measure of antibody titre. The assay is simple to perform, uses readily available reagents and gives comparable sensitivity to rosetting assays. With these features, and the capacity for handling large numbers of trays quickly, this method has obvious advantages in screening for antibody activity in culture supernatants of hybridoma clones.