Using Real-Time PCR Fluorescence Reaction Values to Improve SARS-CoV-2 Virus Detection and Benefit Clinical Decision-Making.

This study aimed to compare the SARS-CoV-2 nucleic acid detection results of the BD MAX™ System and other platforms to formulate an optimized laboratory verification process. The re-examination of 400 samples determined as positive by BD MAX™ indicated that the inconsistency rate between BD MAX™ and the other platforms was 65.8%; the inconsistency rate of single-gene-positive results was as high as 99.2%. A receiver operating characteristic curve was drawn for the relative light unit (RLU) values of samples positive for a single gene, and RLU 800 was used as the cutoff. After setting the retest standard as single-gene positive and RLU ≥ 800, the number of the 260 BD MAX™ single-gene positives that needed to be confirmed again was 36 (13.8%) and the number that could be directly reported as negative was 224 (86.2%). This verification process can shorten the reporting period and speed up the epidemic adjustment time and turnover rate of special wards, thereby improving SARS-CoV-2 detection efficiency and clinical decision-making.

Modification and optimization of the inhibition of HIV-1 cell-to-cell transmission assay.

HIV-1 infection is caused by cell-free and cell-associated viruses. Currently most of the assays used to screen potential HIV-1 entry inhibitors focus on the inhibition of cell-free viruses. One assay that is widely employed is the TZM-bl neutralization assay that uses pseudotyped viruses. However, a study by Abela et al. showed that many inhibitors that potently inhibit cell-free HIV-1 in this assay can be less effective against the cell-to-cell transmission of the virus. These researchers then designed a method to screen entry inhibitors for activity against cell-associated HIV-1, using pseudotyped viruses. The main limitation of this method, however, was that it can only be reliably employed against viruses that cannot infect target cells as cell-free virion in the absence of a polycation supplement such as DEAE (diethylaminoethyl). Thus, in the current study we provide modifications to this method that solves the problem and makes it possible to study entry inhibitors against cell-to-cell infection of both polycation depend and independent viruses. The main modification involves the introduction of the relative light unit (RLU) vs. virus producing 293-T cells / corresponding supernatants graph. This graph is used to select a virus input that only allows for the detection of cell-associated viruses infection.•The method is a modification of the cell-to-cell transmission assay published by Abela et al.•The method allows for the study of the inhibition of cell-to-cell transmission of both polycation dependent and independent HIV-1 pseudoviruses.

Detection and characterization of estradiol (E2) and unconjugated estriol (uE3) immunoassay interference due to anti-bovine alkaline phosphatase (ALP) antibodies.

OBJECTIVES: Competitive immunoenyzmatic assays for estradiol (E2) and unconjugated estriol (uE3) on UniCel DxI 800 Access immunoassay systems (Beckman Coulter) utilize bovine alkaline phosphatase (ALP) for amplification. In these assays, rare 'IND' error flags indicate that a relative light unit (RLU) raw result is past the high or low end of the calibration curve but cannot be differentiated from an instrument error or analytical interference. The present studies were conducted to establish a protocol to identify analytical interference and to characterize its mechanism when present. DESIGN AND METHODS: Matrix and recovery studies were conducted to establish a protocol for interference identification. Spiking experiments with inactivated calf intestinal ALP were performed to determine whether interference could be blocked. Commercial anti-ALP antibodies (Abs) were spiked into human serum to model assay interference. Three E2 immunoassays which do not include ALP as a reagent component (cobas e602, Roche; Centaur XP, Siemens; ARCHITECT i2000SR, Abbott) were tested for comparative purposes. RESULTS: 1:2 dilution of specimen into Access Sample Diluent A (Beckman) differentiated IND error flags due to true low results (e.g. less than the analytical measurement range; AMR) from those due to assay interference. Interferences were reduced by pre-incubation with inactivated ALP and could be replicated by spiking with commercial anti-ALP Abs. CONCLUSIONS: Patient anti-bovine ALP Abs can cause interference on DxI 800 E2 and uE3 assays. This model can be used to investigate interference risk with other ALP-dependent assays.

Study of chemiluminescence measured by luminometry and its application in the estimation of postmortem interval of bone remains.

A substantial challenge faced by forensic medicine is determining the postmortem interval (PMI) of skeletonized remains. Currently, the luminol method is of limited forensic usefulness, since it uses qualitative and subjective methods to estimate PMI by the naked eye assessing the degree of chemiluminescence (CL) emitted by bone remains, a technique which is not sensitive enough to distinguish between historical or forensically significant time intervals. The aim of the present study was to use a direct and accurate measurement of the CL by luminol technique in relative light units (RLU) using a luminometer to establish this method as a possible complementary and low cost tool for the determination of the PMI for distinguishing between remains of medical-legal (<20 years) and historical (≥20 years) interest in 102 femur remains with a range of PMI between 15 and 64 years. The results suggest that, under favorable conditions, the luminol technique can detect haemoglobin in the bone in a PMI range of 0-65 years, finding significant differences in the CL intensity among samples with PMI < 20 years and PMI ≥ 20 years. In addition, the intensities of CL measured at 10 s, 15 s and 20 s after reaction with luminol show a statistically significant inverse relationship with PMI in the bone studied, following a decreasing logarithmic model. The conclusion is that this quantitative, objective and contrastable technique could be very useful for determining the PMI in bone remains, since it allows a good degree of precision and eliminates the subjectivity introduced by qualitative techniques.

Residual contamination and bioburden after reprocessing of single-use endoscopic ultrasound needles: An ex vivo study.

BACKGROUND AND AIM: Endoscopic ultrasound (EUS) aspiration needles are single-use devices. However, in many centers, because of cost-constraints, these devices are reused multiple times. We studied microbiological contamination and bioburden on reprocessed needles to evaluate whether these devices can be successfully sterilized. METHODS: We studied 10 EUS needles each of 19 G, 22 G, and 25 G in size, and five 22-G ProCore needles. After initial use, each needle was reprocessed by a standardized protocol. We used standard microbiological cultures, as well as ATP bioluminescence technique to quantify bioburden as relative light units (RLU). We defined significant soil contamination by RLU values >200. We also used extractant fluid to disrupt cell membranes in an attempt to enhance ATP detection. RESULTS: We found culture positivity in 3/34 (8.8%), and detectable bioburden on the exposed surface of 33/35 (94.3%), and inside lumen of 29 (82.9%) reprocessed FNA needles. Significant bioburden was found in three (8.6%) and two (5.7%) needles on the surface and lumen, respectively. We found that use of extractant fluid enhanced detection of bioburden. Larger (19 G) needles had higher surface contamination (P = 0.016), but there was no relation of luminal contamination with needle diameter (P = 0.138). Sheath design and presence of side bevel did not influence extent of contamination. There was significant correlation between the surface and intraluminal bioburden (P < 0.001). CONCLUSIONS: There is significant bioburden in reprocessed EUS needles; standard microbiological cultures have low sensitivity for detection of needle contamination. We have provided objective evidence for the futility of reprocessing attempts, and practice of EUS needle reuse should be discontinued.

Analysis by a highly sensitive split luciferase assay of the regions involved in APP dimerization and its impact on processing.

Alzheimer's disease (AD) is a neurodegenerative disease that causes progressive loss of cognitive functions, leading to dementia. Two types of lesions are found in AD brains: neurofibrillary tangles and senile plaques. The latter are composed mainly of the ß-amyloid peptide (Aß) generated by amyloidogenic processing of the amyloid precursor protein (APP). Several studies have suggested that dimerization of APP is closely linked to Aß production. Nevertheless, the mechanisms controlling APP dimerization and their role in APP function are not known. Here we used a new luciferase complementation assay to analyze APP dimerization and unravel the involvement of its three major domains: the ectodomain, the transmembrane domain and the intracellular domain. Our results indicate that within cells full-length APP dimerizes more than its α and ß C-terminal fragments, confirming the pivotal role of the ectodomain in this process. Dimerization of the APP transmembrane (TM) domain has been reported to regulate processing at the γ-cleavage site. We show that both non-familial and familial AD mutations in the TM GXXXG motifs strongly modulate Aß production, but do not consistently change dimerization of the C-terminal fragments. Finally, we found for the first time that removal of intracellular domain strongly increases APP dimerization. Increased APP dimerization is linked to increased non-amyloidogenic processing.

Transcriptional regulation of microRNA-100, -146a, and -150 genes by p53 and NFκB p65/RelA in mouse striatal STHdh(Q7)/ Hdh(Q7) cells and human cervical carcinoma HeLa cells.

MicroRNA (miRNA) genes generally share many features common to those of protein coding genes. Various transcription factors (TFs) and co-regulators are also known to regulate miRNA genes. Here we identify novel p53 and NFκB p65/RelA responsive miRNAs and demonstrate that these 2 TFs bind to the regulatory sequences of miR-100, -146a and -150 in both mouse striatal and human cervical carcinoma cells and regulate their expression. p53 represses the miRNAs while NFκB p65/RelA induces them. Further, we provide evidence that exogenous p53 inhibits NFκB p65/RelA activity by reducing its nuclear content and competing with it for CBP binding. This suggests for the existence of a functional cross-talk between the 2 TFs in regulating miRNA expression. Moreover, promoter occupancy assay reveals that exogenous p53 excludes NFκB p65/RelA from its binding site in the upstream sequence of miR-100 gene thereby causing its repression. Thus, our work identifies novel p53 and NFκB p65/RelA responsive miRNAs in human and mouse and uncovers possible mechanisms of co-regulation of miR-100. It is to be mentioned here that cross-talks between p53 and NFκB p65/RelA have been observed to define the outcome of several biological processes and that the pro-apoptotic effect of p53 and the pro-survival functions of NFκB can be largely mediated via the biological roles of the miRNAs these TFs regulate. Our observation with cell lines thus provides an important platform upon which further work is to be done to establish the biological significance of such co-regulation of miRNAs by p53 and NFκB p65/RelA.

A comparative study of two different assay kits for the detection of secreted alkaline phosphatase in HPV antibody neutralization assays.

To assess immunogenicity and development of antibodies in the context of vaccination, it is critical to quantify titers of neutralizing antibodies. We have been employing the 293TT cell-based neutralization assay system to quantify anti-HPV neutralizing antibodies. In this system, human papillomavirus (HPV) pseudovirion (PsV) particles encapsidating secreted alkaline phosphatase (SEAP) gene are used to measure infection of 293TT cells in 72-hr cell-culture supernatants. SEAP has traditionally been measured by Great EscAPe™ SEAP Chemiluminescence Kit 2.0 (GE). To reduce the cost, and to potentially increase efficiency, we sought a cheaper kit with better detection capability. Performance characteristics of the newer chemiluminescence kit, ZiVa® Ultra SEAP Plus Assay (Ziva) and GE were compared using the 293TT system. Dose titration of HPV PsV 16 or 18 showed that signal-to-noise ratios at 48 and 72 hr post-infection were higher for ZiVa at nearly all doses. ZiVa was superior to GE as it was able to detect SEAP at 48 hr, as well as when lower numbers of 293TT cells were used. The ability of ZiVa to quantitate HPV-16 and -18 neutralizing antibody titers was tested using sera from Cervarix® immunized individuals. Spearman rank correlational analyses showed excellent correlations between the titers obtained with ZiVa and GE for anti-HPV16 (r = 0.9822, p < 0.0001) and anti-HPV18 (r = 0.9832, p < 0.0001) antibodies. We concluded that ZiVa is superior to GE in detecting SEAP, and the antibody titers in sera of vaccinated individuals were similar to those obtained with GE. Thus, Ziva is a suitable alternative to GE.

Comparison of hybrid capture 2 High-Risk HPV results in the low positive range with cobas® HPV Test results from the ATHENA study.

BACKGROUND: The increasing importance of high-risk human papillomavirus (hrHPV) testing in cervical cancer screening warrants evaluation of HPV DNA tests with an equivocal zone requiring retesting of samples in the low positive range. OBJECTIVES: To compare the results of the digene hc2 High Risk HPV DNA Test (hc2), which has a manufacturer's recommended retesting zone with the cobas HPV Test, a real-time polymerase chain reaction amplification test without an equivocal range. STUDY DESIGN: A retrospective subanalysis of the ATHENA study comparing results for hc2 High Risk HPV DNA Test and the cobas HPV Test using the LINEAR ARRAY HPV Genotyping Test (LA) and Sanger sequencing as comparators was performed. The ability of each test to detect high-grade cervical disease in the equivocal range was also evaluated. RESULTS: 5.2% of samples fell within the equivocal zone (RLU/CO 1.0-2.5) and required retesting with the hc2 High Risk HPV DNA Test. In this low-positive range the cobas HPV Test showed better positive percent agreement (PPA) than hc2 High Risk HPV DNA Test for LA and sequencing (84.2% vs.70.9% and 92.1% vs.82.5%, respectively). hc2 High Risk HPV DNA Test and the cobas HPV Test demonstrated comparable sensitivity for detection of high-grade disease in the equivocal range. In the low cobas HPV Test range (cycle threshold [Ct] 40-35), the cobas HPV test again demonstrated a better PPA than hc2 High Risk HPV DNA Test with LA and sequencing as comparators and more high-grade disease was detected by the cobas HPV Test than hc2 High Risk HPV DNA Test. CONCLUSION: The cobas HPV Test demonstrates reliable performance in the hc2 High Risk HPV DNA Test equivocal zone, thus supporting it as an option for HPV testing that avoids the need for retesting.

Nanoparticle-DNA-polymer composites for hepatocellular carcinoma cell labeling, sensing, and magnetic resonance imaging.

This paper describes comparative studies and protocols in (1) self-assembling of ultrasmall superparamagnetic iron oxide nanoparticle (NP), circular plasmid DNA, and branched polyethylenimine (PEI) composites; (2) magnetofection; (3) gene delivery, (4) magnetic resonance imaging (MRI), and (5) cytotoxicity of the composites toward hepatocellular carcinoma HepG2 cells.

Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers.

DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 µg/cm(2).h and 0.385 µg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.