scholarly journals Multicenter Evaluation of Two Next-Generation HIV-1 Quantitation Assays, Aptima Quant Dx and Cobas 6800, in Comparison to the RealTime HIV-1 Reference Assay

2018 ◽  
Vol 56 (10) ◽  
Author(s):  
Frank Wiesmann ◽  
Robert Ehret ◽  
Gudrun Naeth ◽  
Martin Däumer ◽  
Jörg Fuhrmann ◽  
...  
Keyword(s):  
High Precision ◽  
Good Alternative ◽  
Clinical Samples ◽  
Hiv Viral Load ◽  
Accuracy And Precision ◽  
Low Concentrations ◽  
High Concordance ◽  
Assay Precision ◽  
Hiv 1 ◽  
Native Plasma

ABSTRACT High accuracy and precision at the lower end of quantification are crucial requirements of a modern HIV viral load (VL) assay, since some clinically relevant thresholds are located at 50 and 200 copies/ml. In this study, we compared the performance of two new fully automated HIV-1 VL assays, Aptima HIV-1 Quant Dx and Cobas HIV-1 (Cobas 6800), with the established RealTime m2000 assay. Assay precision and accuracy were evaluated in a retrospective evaluation out of excess plasma material from four HIV-1+ individuals (subtypes B, C, CRF01_AE, and CRF02_AG). Native plasma samples were diluted to nominal concentrations at 50 and 200 copies/ml (according to the RealTime m2000 assay). All dilutions were tested in triplicate in five independent runs over 5 days and in three labs per system. Assay concordance was determined using 1,011 surplus clinical routine samples, as well as selected retrospective longitudinal samples from 7 patients on treatment. The three assays yielded highly concordant results for individual clinical samples (R2 > 0.98; average difference, ≤0.2 log copies/ml) and retrospective longitudinal samples from patients on treatment. The Aptima and RealTime assays showed similar high precision, meeting the 5σ criterion for the majority of samples across all labs and subtypes. The Cobas assay was less precise, missing the 5σ criterion for the majority of samples at low concentrations. In this analysis, results from the Cobas assay appeared less reliable near the clinically relevant cutoff and should be interpreted with more caution in this context. Due to high precision, full automation, and high concordance with the RealTime assay, the Aptima assay represents a good alternative in routine VL monitoring.

DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

2020 ◽  
Author(s):  
M.A. Tyumentseva ◽  
◽  
A.I. Tyumentsev ◽  
V.G. Akimkin ◽  
◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Health Monitoring ◽  
Biological Samples ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Low Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

Additional HIV-1 mutation patterns associated with reduced phenotypic susceptibility to etravirine in clinical samples

AIDS ◽  
2009 ◽  
Vol 23 (12) ◽  
pp. 1602-1605 ◽  
Author(s):  
Ron M Kagan ◽  
Prakash Sista ◽  
Theresa Pattery ◽  
Lee Bacheler ◽  
Dale A Schwab
Keyword(s):  
Clinical Samples ◽  
Hiv 1 ◽  
Phenotypic Susceptibility

Simultaneous quantification of vitamin E and vitamin E metabolites in equine plasma and serum using LC-MS/MS

2021 ◽  
pp. 104063872110054
Author(s):  
Hadi Habib ◽  
Carrie J. Finno ◽  
Ingrid Gennity ◽  
Gianna Favro ◽  
Erin Hales ◽  
...  
Keyword(s):  
Vitamin E ◽  
Normal Growth ◽  
Alpha Tocopherol ◽  
Limits Of Detection ◽  
Phase Gradient ◽  
Equine Plasma ◽  
Accuracy And Precision ◽  
Low Concentrations ◽  
Liquid Chromatography Tandem Mass ◽  
Acquity Uplc

Vitamin E deficiencies can impact normal growth and development in humans and animals, and assessment of circulating levels of vitamin E and its metabolites may be an important endpoint for evaluation. Development of a sensitive method to detect and quantify low concentrations of vitamin E and metabolites in biological specimens allows for a proper diagnosis for patients and animals that are deficient. We developed a method to simultaneously extract, detect, and quantify the vitamin E compounds alpha-tocopherol (α-TP), gamma-tocopherol (γ-TP), alpha-tocotrienol (α-TT), and gamma-tocotrienol (γ-TT), and the corresponding metabolites formed after β-oxidation of α-TP and γ-TP, alpha-carboxymethylbutyl hydroxychroman (α-CMBHC) and alpha- or gamma-carboxyethyl hydroxychroman (α- or γ-CEHC), respectively, from equine plasma and serum. Quantification was achieved through liquid chromatography–tandem mass spectrometry. We applied a 96-well high-throughput format using a Phenomenex Phree plate to analyze plasma and serum. Compounds were separated by using a Waters ACQUITY UPLC BEH C18 column with a reverse-phase gradient. The limits of detection for the metabolites and vitamin E compounds were 8–330 pg/mL. To validate the method, intra-day and inter-day accuracy and precision were evaluated along with limits of detection and quantification. The method was then applied to determine concentrations of these analytes in plasma and serum of horses. Alpha-TP levels were 3–6 µg/mL of matrix; the metabolites were found at much lower levels, 0.2–1.0 ng/mL of matrix.

scholarly journals Performance of 2 Polymerization Protocols Targeting Cloned Toxoplasma Parasites

2018 ◽  
Vol 6 (9) ◽  
pp. 1577-1580
Author(s):  
Nihal A. Hanafy ◽  
Mohamed S. Badr ◽  
Ghada M. Nasr
Keyword(s):  
Toxoplasma Gondii ◽  
Real Time ◽  
Parasitic Infection ◽  
Clinical Samples ◽  
Molecular Assays ◽  
Low Concentrations ◽  
Target Dna ◽  
Primer Sets ◽  
Amplification Failure ◽  
Reference Samples

BACKGROUND: Toxoplasma gondii is a common parasitic infection of humans. Infection is usually mild. Serious complications can occur in pregnant and immunocompromised patients. AIM: The present study aims to investigate the performance of 2 different PCR protocols; real-time quantitative molecular assays (qPCR) and conventional molecular assays (cPCR), using 2 different sets of primers and by using cloned purified Toxoplasma genomic substances to be evaluated as reference samples. METHODS: The target DNA was provided in 8 different quantities. RESULTS: Amplification failure was reported only with the cPCR in samples of low concentrations using both primer sets. Quantitative PCR detected the 8 different dilutions of the purified Toxoplasma gondii using the 2 sets of primers while cPCR was sensitive to detect only 6 different dilutions. CONCLUSION: Generally real-time quantitative molecular assays, is easy to use method compared to conventional PCR assay and produces more reliable results within only one hour time but still the possible application of qPCRs in routine diagnosis necessitates analysis of a large number of clinical samples in further studies to make the proper choice.

scholarly journals The evolution of regulatory elements in the emerging promoter variant strains of HIV-1

2021 ◽  
Author(s):  
Disha Bhange ◽  
Nityanand Prasad ◽  
Swati Singh ◽  
Harshit Kumar Prajapati ◽  
Shesh Prakash Maurya ◽  
...  
Keyword(s):  
Viral Latency ◽  
Regulatory Elements ◽  
The Other ◽  
Clinical Samples ◽  
Latent Reservoir ◽  
Viral Gene ◽  
Viral Promoter ◽  
Reservoir Characteristics ◽  
Hiv 1

AbstractIn a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naïve subjects, we identified nine different promoter-variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1α/LEF-1, RBEIII, AP-1, and NF-κB, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but not significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a co-infection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time-points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy.Significance StatementA unique conglomeration of TFBS enables the HIV-1 promoter to accomplish two diametrically opposite functions – transcriptional activation and transcriptional silencing. The various phases of viral latency - establishment, maintenance, and reversal - collectively determine the replication fitness of individual viral strains. A profound variation in the TFBS composition of the viral promoter may significantly alter the viral latency properties and the latent reservoir characteristics. Although the duplication of certain TFBS remains a quality unique to HIV-1C, the high-level genetic recombination of HIV-1 may promote the transfer of such molecular properties to the other HIV-1 subtypes. The emergence of several promoter-variant viral strains may make the task of a ‘functional cure’ more challenging in HIV-1C.

Improved XLT4 Agar: Small Addition of Peptone to Promote Stronger Production of Hydrogen-Sulfide by Salmonellae

1994 ◽  
Vol 57 (10) ◽  
pp. 854-858 ◽  
Author(s):  
R. G. MILLER ◽  
C. R. TATE ◽  
E. T. MALLINSON
Keyword(s):  
Hydrogen Sulfide ◽  
Clinical Samples ◽  
Pork Sausage ◽  
Bacterial Cultures ◽  
Proteose Peptone ◽  
Low Concentrations ◽  
Production Of Hydrogen ◽  
Hydrogen Sulfide Production ◽  
Selective Plating ◽  
Pure Bacterial Cultures

Xylose lysine tergitol4 agar (XLT4) is a highly selective plating medium used for isolating salmonellae. Studies have shown that XLT4 increases the recovery of salmonellae found in food, environmental and clinical samples. Further testing demonstrated that the addition of low concentrations of proteose peptone No. 3 (pp3) to XLT4 produced blacker Salmonella colonies in shorter incubation times (increased hydrogen sulfide production), while still maintaining strong inhibition of competing bacteria. The increased black colony formation facilitates prompt recognition of the weaker hydrogen sulfide-producing Salmonella strains. Test concentrations of pp3 at 0.5, 1.2 and 1.8 g/L were added to XLT4 and compared with plain XLT4 using pure bacterial cultures. In addition, these four plating media, plus xylose lysine desoxycholate agar (XLD) were evaluated using nonspiked chicken liver and pork sausage samples. The concentration of 1.2 g/L of pp3 in XLT4 gave the best overall results. In virtually all cases, the Salmonella colonies were larger and more black than on plain XLT4 without pp3. The improved XLT4 is recommended for more reliable detection of salmonellae from food, environmental and clinical samples.

Comparison of the Abbott Alinity m and m2000 assays for the quantification of HIV-1, HCV and HBV in clinical samples

2020 ◽  
Vol 126 ◽  
pp. 104331
Author(s):  
Lina Mouna ◽  
Coralie Pallier ◽  
Stéphanie Proust ◽  
Corinne Prégermain ◽  
Anne-Marie Roque-Afonso
Keyword(s):  
Clinical Samples ◽  
Hiv 1

scholarly journals Performance of a high-throughput next-generation sequencing method for analysis of HIV drug resistance and viral load

2020 ◽  
Vol 75 (12) ◽  
pp. 3510-3516 ◽  
Author(s):  
Jessica M Fogel ◽  
David Bonsall ◽  
Vanessa Cummings ◽  
Rory Bowden ◽  
Tanya Golubchik ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Viral Load ◽  
Next Generation Sequencing ◽  
High Throughput ◽  
Whole Genome ◽  
Hiv Viral Load ◽  
Viral Loads ◽  
Viral Load Assay ◽  
Generation Sequencing ◽  
Hiv 1

Abstract Objectives To evaluate the performance of a high-throughput research assay for HIV drug resistance testing based on whole genome next-generation sequencing (NGS) that also quantifies HIV viral load. Methods Plasma samples (n = 145) were obtained from HIV-positive MSM (HPTN 078). Samples were analysed using clinical assays (the ViroSeq HIV-1 Genotyping System and the Abbott RealTime HIV-1 Viral Load assay) and a research assay based on whole-genome NGS (veSEQ-HIV). Results HIV protease and reverse transcriptase sequences (n = 142) and integrase sequences (n = 138) were obtained using ViroSeq. Sequences from all three regions were obtained for 100 (70.4%) of the 142 samples using veSEQ-HIV; results were obtained more frequently for samples with higher viral loads (93.5% for 93 samples with >5000 copies/mL; 50.0% for 26 samples with 1000–5000 copies/mL; 0% for 23 samples with <1000 copies/mL). For samples with results from both methods, drug resistance mutations (DRMs) were detected in 33 samples using ViroSeq and 42 samples using veSEQ-HIV (detection threshold: 5.0%). Overall, 146 major DRMs were detected; 107 were detected by both methods, 37 were detected by veSEQ-HIV only (frequency range: 5.0%–30.6%) and two were detected by ViroSeq only. HIV viral loads estimated by veSEQ-HIV strongly correlated with results from the Abbott RealTime Viral Load assay (R2 = 0.85; n = 142). Conclusions The NGS-based veSEQ-HIV method provided results for most samples with higher viral loads, was accurate for detecting major DRMs, and detected mutations at lower levels compared with a method based on population sequencing. The veSEQ-HIV method also provided HIV viral load data.

scholarly journals Analysis for Alpha-Pyrrolidinovalerophenone and Its 2-Oxo-PVP Metabolite in Plasma by Liquid Chromatography–Tandem Mass Spectrometry

2019 ◽  
Author(s):  
David M Andrenyak ◽  
David E Moody ◽  
Jonathan M Crites ◽  
Michael H Baumann
Keyword(s):  
Room Temperature ◽  
Rat Plasma ◽  
Sample Volume ◽  
Internal Standards ◽  
Accuracy And Precision ◽  
Assay Performance ◽  
Liquid Chromatography Tandem Mass ◽  
Assay Precision ◽  
Recreational Use ◽  
Positive Ion

Abstract Alpha-pyrrolidinovalerophenone (alpha-PVP), a novel psychoactive substance, has widespread recreational use. This with interest in its pharmacological effects creates a need for methods that measure alpha-PVP concentrations. We therefore developed a LC-MS/MS method that can quantitate alpha-PVP and 2-oxo-PVP in rat plasma using a 0.1-mL sample volume. Addition of internal standards (2.5 ng/mL alpha-PVP-d8/2-oxo-PVP-d6) was followed by liquid–liquid extraction with 1-chlorobutane:acetonitrile (4:1), evaporation and reconstitution with 0.1% formic acid. Extracts were analyzed by LC-MS/MS using an Agilent 1100 HPLC and a Thermo Scientific TSQ Quantum Access MS/MS, with a YMC ODS-AQ, 50 mm × 2 mm, 3 μm column. The mobile phase was 0.1% formic acid:acetonitrile gradient at a 0.2-mL/minute flow rate with positive ion electrospray. SRM was used for the analysis with transitions: alpha-PVP, 232 → 91; alpha-PVP-d8, 240 → 91; 2-oxo-PVP, 246 → 91; 2-oxo-PVP-d6, 252 → 91. Alpha-PVP and 2-oxo-PVP eluted at 6.4 and 8.9 min. Calibrators range from 0.25 to 500 ng/mL. Accuracy and precision evaluated quality control samples prepared at 0.75, 10 and 400 ng/mL. The intra-assay evaluation also included the 0.25-ng/mL LOQs prepared in six different blank plasma sources. The intra-assay accuracy ranged from 88.9 to 117.8% of the target, and the intra-assay precision ranged from 0.9 to 16.0%. The inter-assay accuracy ranged from 98.7 to 110.7% of the target, and the inter-assay precision ranged from 4.5 to 12.0%. Extraction recovery was at least 52% for alpha-PVP and 67% for 2-oxo-PVP. Ionization recoveries were at least 64% for alpha-PVP and 82% for 2-oxo-PVP. These losses did not adversely affect assay performance. Alpha-PVP and 2-oxo-PVP controls were stable at room temperature for up to 24 h and frozen for at least 36 days. Alpha-PVP and 2-oxo-PVP were also stable in processed samples (extracts) stored at room temperature for at least 24 days. The procedure was used to analyze rat plasma samples from a pharmacokinetic study.

Sign in / Sign up

Export Citation Format

Share Document