scholarly journals Clinical Validation of a Quantitative HIV-1 DNA Droplet Digital PCR assay: Applications for Detecting Occult HIV-1 Infection and Monitoring Cell-associated HIV-1 Dynamics across Different Subtypes in HIV-1 Prevention and Cure Trials

2020 ◽  
Author(s):  
Laura Powell ◽  
Adit Dhummakupt ◽  
Lilly Siems ◽  
Dolly Singh ◽  
Yann Le Duff ◽  
...  
Keyword(s):  
Mononuclear Cells ◽  
Limit Of Detection ◽  
Single Copy ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Analytical Sensitivity ◽  
Antibody Testing ◽  
Viral Reservoirs ◽  
Peripheral Blood Mononuclear ◽  
Hiv 1

Background: In HIV-1-exposed infants, nucleic acid testing (NAT) is required to diagnose infection since passively transferred maternal antibodies preclude antibody testing. The sensitivity of clinical NAT assays is lowered with infant antiretroviral prophylaxis and, with empiric very early antiretroviral treatment of high-risk infants, thereby impacting early infant diagnosis. Similarly, adult HIV-1 infections acquired under pre-exposure prophylaxis may occur at low levels (occult infection), with undetectable plasma viremia and indeterminate antibody tests, for which HIV-1 DNA testing maybe a useful adjunct. Cell-associated HIV-1 DNA concentrations are also used to monitor HIV-1 persistence in viral reservoirs with relevance to HIV-1 cure therapeutics, particularly in perinatal infections. Methods: The analytical sensitivity and specificity of an HIV-1 DNA droplet digital PCR (ddPCR) assay was determined, across different HIV-1 subtypes, using serial dilutions of a plasmid containing a 160-base pair sequence of the HIV-1 LTR-gag spiked into peripheral blood mononuclear cells (PBMCs), with MOLT-4 cells or PBMCs infected with different HIV-1 subtypes (A, B and C), and U1 cells spiked into PBMCs. Inter- and intra-run variability were used to determine assay precision. Results: The HIV-1 LTR-gag ddPCR assay was reliable and reproducible, and exhibited high analytical specificity with sensitivity to near single copy level, across multiple HIV-1 subtypes, and a limit of detection of 4.09 copies/million PBMCs. Conclusions. This assay has applications for detecting occult HIV-1-infection that may occur in the setting of combination and long-acting regimens used for HIV-1 prevention, across different HIV-1 subtypes, in both infants and adults, and in HIV-1 cure interventions, particularly with perinatal infections.

Novel Phorbol Esters Exert Dichotomous Effects on Inhibition of HIV-1 Infection and Activation of Latent HIV-1 Expression

2005 ◽  
Vol 16 (5) ◽  
pp. 303-313
Author(s):  
Yu Zhong ◽  
Yuji Matsuya ◽  
Hideo Nemoto ◽  
Masao Mori ◽  
Haruo Saito ◽  
...  
Keyword(s):  
Mtt Assay ◽  
Mononuclear Cells ◽  
Phorbol Esters ◽  
High Concentration ◽  
Viral Reservoirs ◽  
Peripheral Blood Mononuclear ◽  
Latently Infected ◽  
Low Cytotoxicity ◽  
Latent Hiv ◽  
Hiv 1

Two new phorbol esters, NPB-11 (12- O-methoxymethylphorbol-13-decanoate) and NPB-15 (12- O-benzyloxymethylphorbol-13-decanoate) were synthesized. The compounds exhibited potent anti-HIV-1 activity and low cytotoxicity in MT-4 cells by MTT assay even at a high concentration [50% cytotoxic concentrations (CC50) were 8.32 and 4.39 μg/ml, respectively]. Two inhibitors strongly suppressed HIV-1 (IIIB strain) replication in MT-4 cells with a 50% effective concentration (EC50) of 1.3 and 0.27 ng/ml, respectively. NPB-11 efficiently blocked replication of both X4 and R5 HIV-1 in PHA-activated peripheral blood mononuclear cells and MT-4 cells as revealed by p24 assay. The antiviral activity appeared to be mediated, at least partially, by the down-regulation of the expression of CD4 and the HIV-1 co-receptors, CXCR4 and CCR5. The compounds were also capable of selectively up-regulating HIV-1 expression in a variety of latently infected cell lines and inducing cell death in HIV-1 infected cells. The effect of NPBs on the induction of HIV-1 was specifically blocked by nontoxic doses of a protein kinase C blocker, staurosporine. NPB-11 blocked the spread of HIV-1 released from latently infected ACH-2 cells to MT-4 cells in a co-culture system. When combined with AZT, NPB-11 synergistically inhibited HIV-1 replication in MTT assay using MT-4 cells. These data suggest that these agents might be useful in reducing persistent viral reservoirs in patients and as adjuvant therapy in patients treated with HAART.

scholarly journals Rapid Detection and Quantification of Mycobacterium tuberculosis DNA in Paraffinized Samples by Droplet Digital PCR: A Preliminary Study

2021 ◽  
Vol 12 ◽  
Author(s):  
Maria Antonello ◽  
Rossana Scutari ◽  
Calogero Lauricella ◽  
Silvia Renica ◽  
Valentina Motta ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Bacterial Load ◽  
Reference Method ◽  
Digital Pcr ◽  
Probit Analysis ◽  
Droplet Digital Pcr ◽  
Smear Microscopy ◽  
Analytical Sensitivity ◽  
Diagnostic Challenge ◽  
Culture Positive

Background: Rapid and reliable diagnosis of tuberculosis (TB) represents a diagnostic challenge in compartmentalized extrapulmonary TB infection because of the small number of mycobacteria (MTB) and the frequent lack of fresh samples to perform culture. Here, we estimate the performances of homemade droplet digital PCR (ddPCR)-based assays against culture in 89 biopsies, for those fresh and formalin-fixed and paraffin-embedded (FFPE) subsamples were available.Methods: MTB diagnosis in fresh subsamples was performed by culture. Fresh subsamples were also analyzed for acid-fast bacilli smear-microscopy (AFB) and Xpert® MTB/RIF (Xpert). MTB examination was repeated in blind in the 89 FFPE subsamples by in-house ddPCR assays targeting the IS6110 and rpoB. Analytical sensitivity of ddPCR assays was evaluated using serial dilution of H37Rv strain. Limit of detection (LOD) was calculated by probit analysis. Results were expressed in copies/106 cells.Results: IS6110 and rpoB ddPCR assays showed a good linear correlation between expected and observed values (R2: 0.9907 and 0.9743, respectively). Probit analyses predicted a LOD of 17 and 40 copies/106 cells of MTB DNA for IS6110 and rpoB, respectively. Of the 89 biopsies, 68 were culture positive and 21 were culture negative. Considering mycobacterial culture as reference method, IS6110 assay yielded positive results in 67/68 culture-positive samples with a median interquartile range (IQR) of 1,680 (550–8,444) copies/106 cells (sensitivity: 98.5%; accuracy: 98.9). These performances were superior to those reported by the rpoB assay in FFPE subsamples (sensitivity: 66.20%; accuracy: 74.1) and even superior to those reported by Xpert and AFB in fresh subsamples (sensitivity: 79.4 and 33.8%, respectively; accuracy: 84.3 and 49.4, respectively). When Xpert and AFB results were stratified according to mycobacterial load detected by rpoB and IS6110 ddPCR, bacterial load was lower in Xpert and AFB negative with respect to Xpert and AFB-positive samples (p = 0.003 and 0.01 for rpoB and p = 0.01 and 0.11 for IS6110), confirming the poor sensitivity of these methods in paucibacillary disease.Conclusion: ddPCR provides highly sensitive, accurate, and rapid MTB diagnosis in FFPE samples, as defined by the high concordance between IS6110 assay and culture results. This approach can be safely introduced in clinical routine to accelerate MTB diagnosis mainly when culture results remain unavailable.

scholarly journals The Range of Human APOBEC3H Sensitivity to Lentiviral Vif Proteins

2009 ◽  
Vol 84 (1) ◽  
pp. 88-95 ◽  
Author(s):  
Melody M. H. Li ◽  
Lily I. Wu ◽  
Michael Emerman
Keyword(s):  
Antiviral Activity ◽  
Mononuclear Cells ◽  
Limit Of Detection ◽  
Human Peripheral Blood ◽  
Peripheral Blood Mononuclear ◽  
Endogenous Protein ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Human Apobec3g

ABSTRACT The APOBEC3H gene is polymorphic in humans, with four major population-dependent haplotypes that encode proteins with different levels of antiviral activity. Haplotype II, present most frequently in African populations, encodes the most stable protein and is most active against human immunodeficiency virus type 1 (HIV-1). In contrast to human APOBEC3G, which can be completely counteracted by HIV-1 Vif, the protein encoded by APOBEC3H haplotype II is only partially sensitive to Vif, while the protein encoded by APOBEC3H haplotype I is completely resistant to HIV-1 Vif. We mapped a residue on APOBEC3H that determines this partial Vif sensitivity. However, it is unclear how HIV-1 can replicate in vivo without the ability to neutralize APOBEC3H antiviral activity. In order to directly address this question, we cloned vif genes from HIV-1-infected individuals with different APOBEC3H genotypes and tested them for their ability to inhibit human APOBEC3H. We found that while the APOBEC3H genotype of infected individuals significantly influences the activity of Vif encoded by their virus, none of the Vif variants tested can completely neutralize APOBEC3H as well as they neutralize APOBEC3G. Consistent with this genetic result, APOBEC3H protein expression in human peripheral blood mononuclear cells was below our limit of detection using newly developed antibodies against the endogenous protein. These results demonstrate that human APOBEC3H is not as strong of a selective force for current HIV-1 infections as human APOBEC3G.

scholarly journals A Novel Primer Probe Set for Detection of SARS-CoV-2 by Sensitive Droplet Digital PCR

2020 ◽  
Author(s):  
Fang Wang ◽  
Umar Pervaiz ◽  
Hongwei Tian ◽  
Algahdary Omar Ahmed Omar Mariam ◽  
Mahasin Abdallah Mohammed Hamid ◽  
...  
Keyword(s):  
Lower Limit ◽  
Detection Rate ◽  
Limit Of Detection ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
N Gene ◽  
Analytical Sensitivity ◽  
Ct Value ◽  
The Mean ◽  
Probe Set

AbstractBackgroundThe current increase in the spread of (SARS-CoV-2) critically needs a multitarget diagnostic assays to promote analytical sensitivity to facilitate the public health actions.ObjectiveThe aim of this study was to develop a new primer-probe set targeting N gene of SARS-CoV-2 to improve the sensitivity for detection of COVID-19(Corona Virus Disease 2019)in multiplex rRT-PCR (Reversetranscript Realtime PCR) and ddPCR (Droplet Digital PCR).ResultsWe designed primers/probes set N(LZU3) targeting the N gene of 2019-nCov and proved its sensitivity in both rRT-PCR and ddPCR. When the quantity of template was 105 copies/reaction, the mean Ct value of N(LZU3) was 32.563, the detection rate was 91.7%. If the quantity of template was 52.5 copies/reaction, the mean Ct value of N(LZU3) was 33.835, and the detection rate was 83.3%, which were similar with that of N(CDC) and N(USA). The calculated lower limit of detection (LOD) of the new primer-probe set N(LZU3) used in rRT-PCR was 118 copies/reaction. We also did one-step ddPCR for detection the same serial dilution of RNA template. It shows good linearity for primer/probe sets N(LZU3). The calculated lower limit of detection (LOD) of N(LZU3) was 22.4 copies/reaction, which was 1.12 copies/ul.ConclusionThe novel primer-probe set(LZU3) targeting N gene of SARS-CoV-2 could be both used in rRT-PCR and ddPCR with better sensitivity, furthermore, ddPCR method had higer sensitivity than rRT-PCR, hence it could significantly improve SARS-CoV-2 detection efficiency in low virus load and asymptomatic infection.

scholarly journals Inhibition of Human Immunodeficiency Virus Replication by a Dual CCR5/CXCR4 Antagonist

2004 ◽  
Vol 78 (23) ◽  
pp. 12996-13006 ◽  
Author(s):  
Katrien Princen ◽  
Sigrid Hatse ◽  
Kurt Vermeire ◽  
Stefano Aquaro ◽  
Erik De Clercq ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Viral Entry ◽  
Mononuclear Cells ◽  
Human Immunodeficiency Virus Replication ◽  
Cxcr4 Antagonist ◽  
Peripheral Blood Mononuclear ◽  
Transfected Cells ◽  
Immunodeficiency Virus ◽  
Intracellular Ca ◽  
Hiv 1

ABSTRACT Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC50] ranging from 1.2 to 26.5 μM) in various T-cell lines, CCR5- or CXCR4-transfected cells, peripheral blood mononuclear cells (PBMCs), and monocytes/macrophages. AMD3451 also inhibited R5, R5/X4, and X4 HIV-1 primary clinical isolates in PBMCs (IC50, 1.8 to 7.3 μM). A PCR-based viral entry assay revealed that AMD3451 blocks R5 and X4 HIV-1 infection at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca2+ signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca2+ flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did not interfere with chemokine-induced Ca2+ signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca2+ signaling by itself at concentrations up to 400 μM. In freshly isolated monocytes, AMD3451 inhibited the Ca2+ flux induced by CXCL12 and CCL4 but not that induced by CCL2, CCL3, CCL5, and CCL7. The CXCL12- and CCL3-induced chemotaxis was also dose-dependently inhibited by AMD3451. Furthermore, AMD3451 inhibited CXCL12- and CCL3L1-induced endocytosis in CXCR4- and CCR5-transfected cells. AMD3451, in contrast to the specific CXCR4 antagonist AMD3100, did not inhibit but enhanced the binding of several anti-CXCR4 monoclonal antibodies (such as clone 12G5) at the cell surface, pointing to a different interaction with CXCR4. AMD3451 is the first low-molecular-weight anti-HIV agent with selective HIV coreceptor, CCR5 and CXCR4, interaction.

Evidence for late stage compartmentalization of HIV-1 resistance mutations between lymph node and peripheral blood mononuclear cells

AIDS ◽  
2000 ◽  
Vol 14 (15) ◽  
pp. 2273-2281 ◽  
Author(s):  
Da' N. Haddad ◽  
Christopher Birch ◽  
Tracy Middleton ◽  
Dominic E. Dwyer ◽  
Anthony L. Cunningham ◽  
...  
Keyword(s):  
Lymph Node ◽  
Peripheral Blood Mononuclear Cells ◽  
Late Stage ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Resistance Mutations ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Hiv 1

scholarly journals The Effect of Sulforaphane on Glyoxalase I Expression and Activity in Peripheral Blood Mononuclear Cells

Nutrients ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 1773 ◽  
Author(s):  
Michela Alfarano ◽  
Donato Pastore ◽  
Vincenzo Fogliano ◽  
Casper Schalkwijk ◽  
Teresa Oliviero
Keyword(s):  
Gene Expression ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Limit Of Detection ◽  
Peripheral Blood Mononuclear ◽  
Potential Health ◽  
Age Related ◽  
Blood Mononuclear Cells ◽  
Expression Activity

Studies demonstrate that the potential health-beneficial effect of sulforaphane (SR), a compound formed in broccoli, is the result of a number of mechanisms including upregulation of phase two detoxification enzymes. Recent studies suggest that SR increases expression/activity of glyoxalase 1 (Glo1), an enzyme involved in the degradation of methylglyoxal, is major precursor of advanced glycation end products. Those compounds are associated with diabetes complications and other age-related diseases. In this study, the effect of SR on the expression/activity of Glo1 in peripheral blood mononuclear cells (PBMCs) from 8 healthy volunteers was investigated. PBMCs were isolated and incubated with SR (2.5 μM-concentration achievable by consuming a broccoli portion) for 24 h and 48 h. Glo1 activity/expression, reduced glutathione (GSH), and glutathione-S-transferase gene expression were measured. Glo1 activity was not affected while after 48 h a slight but significant increase of its gene expression (1.03-fold) was observed. GSTP1 expression slightly increased after 24 h incubation (1.08-fold) while the expressions of isoform GSTT2 and GSTM2 were below the limit of detection. GSH sharply decreased, suggesting the formation of GSH-SR adducts that may have an impact SR availability. Those results suggest that a regular exposure to SR by broccoli consumption or SR supplements may enhance Glo1.

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