High capacity clinical SARS-CoV-2 molecular testing using combinatorial pooling.

BACKGROUND: The SARS-CoV-2 pandemic led to unprecedented testing demands, causing major testing delays globally. One strategy used for increasing testing capacity was pooled-testing, using a two-stage technique first introduced during WWII. However, such traditional pooled testing was used in practice only when positivity rates were below 2%. METHODS: Here we report the development, validation and clinical application of P-BEST - a single-stage pooled-testing strategy that was approved for clinical use in Israel. RESULTS: P-BEST is clinically validated using 3636 side-by-side tests and is able to correctly detect all positive samples and accurately estimate their Ct value. Following regulatory approval by the Israeli Ministry of Health, P-BEST was used in 2021 to clinically test 837,138 samples using 270,095 PCR tests - a 3.1fold reduction in the number of tests. This period includes the Alpha and Delta waves, when positivity rates exceeded 10%, rendering traditional pooling non-practical. We also describe a tablet-based solution that allows performing manual single-stage pooling in settings where liquid dispensing robots are not available. CONCLUSIONS: Our data provides a proof-of-concept for large-scale clinical implementation of single-stage pooled-testing for continuous surveillance of multiple pathogens with reduced test costs, and as an important tool for increasing testing efficiency during pandemic outbreaks.

Testing samples for SARS-CoV-2 is usually done on one sample at a time. However, the unprecedented demand for testing during the COVID-19 pandemic led to the adoption of pooled testing strategies, where samples are combined before being tested. This strategy requires two rounds: first, each pool of samples is tested, and then a second testing round is performed on individual samples from positive pools. We developed and implemented a pooling method for SARS-CoV-2 that requires a single round of testing, thus enabling the shorter turnaround times required during a pandemic. The method was approved for clinical use in Israel and was used to successfully test 837,138 clinical samples using fewer than a third of the tests usually required. Our study provides a blueprint for rapid implementation of efficient high-throughput testing in future pandemics.

The Concordance between Mumps and Rubella Sero-Positivity among the Israeli Population in 2015.

Mumps and rubella are vaccine-preventable viral diseases through the measles-mumps-rubella-varicella (MMRV) vaccine, administered at 12 months and again at 6 years. We assessed the sero-prevalence of mumps and rubella, identified factors associated with sero-negativity, and evaluated concordance between mumps and rubella sero-positivity. A national cross-sectional sero-survey was conducted on samples collected in 2015 by the Israel National Sera Bank. Samples were tested for mumps and rubella IgG antibodies using an enzyme-linked immunosorbent assay. Of 3131 samples tested for mumps IgG, 84.8% (95%CI: 83.5-86.0%) were sero-positive. Sero-negativity for mumps was significantly associated with age (high odds ratios observed in infants younger than 4 years and 20-29 years old subjects). Of 3169 samples tested for rubella IgG antibodies, 95.2% (95%CI: 94.4-95.9%) were sero-positive. Rubella sero-negativity was significantly associated with age (high odds ratios observed in children younger than 4 years old and adults older than 30 years), males, Jews, and others. Concordant sero-positivity for both mumps and rubella viruses was observed in 83.9% of the tested samples. The Israeli population was sufficiently protected against rubella but not against mumps. Since both components are administered in the MMRV vaccine simultaneously, the mumps component has a lower uptake than rubella and quicker waning.

Severe Complications Following Maxillary Sinus Augmentation Using Poly L-lactide-co-ε-caprolactone-Coated Bovine Bone: A Retrospective Study.

PURPOSE: To describe the postoperative complications following lateral wall sinus augmentation using (poly L-lactideco-ε-caprolactone; PLCL) and natural polysaccharides polymers-coated bovine bone (PBB). The secondary aims were to examine histologic findings and to propose complication management alternatives. MATERIALS AND METHODS: This retrospective study included 61 subjects who underwent 67 lateral wall sinus augmentation procedures using PBB in the standard protocol. In cases that presented complications, treatment included additional antibiotic therapy, implant removal, or sinus reentry and total removal of the grafting material. In three cases, biopsy specimens were taken from the sinuses, and histologic analyses were performed. RESULTS: The prevalence of postoperative complications was 32.8% (22 of 67 cases) in 18 of the patients (29.5%). The most prevalent symptoms were persistent pain (68.2%), swelling (63.6%), and oroantral fistula (54.5%). Radiographic signs appeared in 45.5% of the complications. A total of 24 implants failed; thus, an overall 80.3% survival rate was established at 19 months. The vast majority of complications (86.4%) were treated eventually with reentry surgery and revealed that the sinus was full with granulation tissue surrounding pieces of a nonossified rubber-like material. In cases where implants were placed, nonosseointegrated implants were surrounded by soft tissue. The sinus was cleaned thoroughly; the graft material remnants were removed together with inflamed parts of the sinus membrane, followed by chlorhexidine and saline lavages. In the biopsy specimens taken from the sinus cavity, there were no histologic features of new bone formation around the grafted material. CONCLUSION: Lateral wall maxillary sinus augmentation using PBB was associated with an acute sinus infection histologic appearance and with a 7-times-higher failure rate compared with previous reports. This serious adverse event suggests that PBB cannot be recommended for maxillary sinus augmentations.

Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern in BNT162b2-mRNA-vaccinated individuals.

The BNT162b2 mRNA vaccine is highly effective against SARS-CoV-2. However, apprehension exists that variants of concern (VOCs) may evade vaccine protection, due to evidence of reduced neutralization of the VOCs B.1.1.7 and B.1.351 by vaccine sera in laboratory assays. We performed a matched cohort study to examine the distribution of VOCs in infections of BNT162b2 mRNA vaccinees from Clalit Health Services (Israel) using viral genomic sequencing, and hypothesized that if vaccine effectiveness against a VOC is reduced, its proportion among breakthrough cases would be higher than in unvaccinated controls. Analyzing 813 viral genome sequences from nasopharyngeal swabs, we showed that vaccinees who tested positive at least 7 days after the second dose were disproportionally infected with B.1.351, compared with controls. Those who tested positive between 2 weeks after the first dose and 6 days after the second dose were disproportionally infected by B.1.1.7. These findings suggest reduced vaccine effectiveness against both VOCs within particular time windows. Our results emphasize the importance of rigorously tracking viral variants, and of increasing vaccination to prevent the spread of VOCs.

Seropositivity of measles antibodies in the Israeli population prior to the nationwide 2018 - 2019 outbreak.

Measles vaccine is administered in Israel as part of the routine childhood immunization program, at ages 1 and 6 years. In this study, we assessed seropositivity of the Israeli population against measles before the onset and propagation of the 2018-2019 measles outbreak. From the Israel Center for Disease Control National Serum Bank, 3,164 samples collected during 2015 were tested for measles antibodies. All the tests were performed using Enzyme-Linked Immunosorbent Assay (ELISA) commercial kit (Enzygnost, Anti-Measles Virus/IgG: Behring, Marburg, Germany). The overall seropositivity rate for measles was 90.7%. The seropositivity rate at 6 months and younger was 48.9%, and decreased to 3.8% among infants aged 6-11 months. Seropositivity increased to 90.7% in the 1-4-year age group, and reached 96.1% for 5-9 year-old children. Our results suggest high immunity in the Israeli population against measles virus, but not high enough to prevent outbreaks because of pockets of specific population groups with low immunization coverage. Infants between ages 6 and 11 months and children younger than 2 years had the lowest seropositivity rates being the age groups with the highest attack rates of measles during the epidemic of 2018. Efforts should be aimed at avoiding any delay in vaccination once a child reaches the age of 1 year and improving immunity levels in children aged 1-4 years.

Dimensional changes in the sinus membrane following maxillary sinus augmentation.

OBJECTIVE: The aim of this retrospective study was to measure the sinus membrane's dimensional changes following maxillary sinus augmentation via a lateral approach, and to examine the variables affecting changes in the membrane's thickness. METHOD AND MATERIALS: Sixty-six sinuses corresponding to 50 patients (15 males and 35 females) who underwent lateral wall maxillary sinus augmentation (34 unilateral and 16 bilateral) were retrospectively evaluated. The sinus membrane thickness was measured on cross-sectional cone beam computed tomography (CBCT) scans which were performed prior to and 9 to 11 months' post maxillary sinus augmentation. The Wilcoxon signed-rank test and the Mann Whitney U test were both used to compare between baseline and postoperative sinus membrane thickness. Pearson correlation tests were used to analyze correlations between graft height and sinus membrane thickness changes. RESULTS: The mean age was 53 ± 4 years (45 to 71 years). A total of 132 CBCT scans were analyzed pre- and postoperatively (n = 66). The mean thickness of the sinus membrane before the procedure was 2.61 ± 3.61 mm, while the mean thickness of the membrane after the procedure was 2.94 ± 3.51 (P > .20). Thin membranes at baseline (< 1.56 mm) thickened by a mean of 2.21 ± 2.34 mm, range -0.413 to 10.62 mm, (P < .0001); thicker membranes (≥ 1.56 mm) lost 1.46 ± 3.96 mm thickness, range -7.8 to 9.31 mm (P < .0001). A moderate negative correlation between the baseline membrane thickness and change in thickness was observed (P < .0001, r = -.52). No correlation was found between the graft height and changes in the sinus membrane thickness. CONCLUSION: Lateral wall maxillary sinus augmentation seems to affect the sinus membrane thickness. These changes are associated with the preoperative thickness of the membrane.

The relationship between serum levels of prolactin and growth hormone in the early postnatal period.

BackgroundIn the neonatal period, the pituitary hormones including prolactin (PRL) and human growth hormone (hGH) are secreted in high amounts due to immature feedback mechanisms. As both hormones are secreted in part by the same somatomammotrophic cells, we investigated their relationship in newborns with respect to sex, gestational week, method of delivery, and anthropometric data.MethodsThe serum levels of PRL and hGH were measured in blood drawn from 225 newborns. The newborn data were extracted from medical records.ResultsA positive correlation was found between log-transformations of PRL and hGH (r=0.17; P=0.01; n=225), with a stronger correlation in newborns whose blood samples were taken more than 2 days after birth (r=0.42; P<0.001; n=130). Log-transformations of the PRL/hGH ratio demonstrated a positive correlation with the gestational week (r=0.39; P<0.001; n=200). Multiple regression analysis showed that 15% of the variance in the logarithm of this ratio is attributed to the gestational week.ConclusionIn newborns, serum PRL and hGH levels show a positive correlation that can be explained by common regulatory factors or a drift phenomenon. A higher gestational week is associated with a higher PRL/hGH ratio. Further studies are needed to look for possible confounders and to determine the PRL-hGH relationship in different conditions.

The seroprevalence of West Nile Virus in Israel: A nationwide cross sectional study.

West Nile Virus (WNV) is endemic in Israel, affecting yearly 40-160 individuals. Israel is located on a central migratory path between Africa and Eurasia and most West Nile Fever (WNF) cases reported in recent years were among residents of the coastal plain. The aim of the study was to evaluate the seroprevalence of WNV among the Israeli population and to assess correlates for WNV infection. A cross-sectional nationwide serologic survey was conducted using 3,145 serum samples collected by the national Israeli serum bank during 2011-2014, representing all age and population groups in Israel. Prevalence rates of WNV IgG antibodies were determined. Logistic regressions models were applied to assess the associations between demographic characteristics and WNV seropositivity. 350 samples were positive to WNV (11.1%; 95%CI: 10.0-12.3%). In the multivariable analysis, there was a significant association between seropositivity and the Arab population group vs. Jews and others (OR = 1.86, 95%CI: 1.37-2.52), the time lived in Israel [50-59 years vs. 0-9 years; OR = 10.80 (95%CI: 1.03-113.46) and ≥60 years vs. 0-9 years; OR = 14.00 (1.32-148.31)] residence area] Coastal Plain, Inland Plain (Shfela) and Great Rift Valley vs. Upper Galilee; OR = 2.24 (95%CI: 1.37-3.65), OR = 2.18 (95%CI: 1.18-4.03), OR = 1.90 (95%CI: 1.10-3.30), respectively [and rural vs. urban settlement (OR = 1.65, 95%CI: 1.26-2.16). People, who reside in the Coastal Plain, Inland Plain and Great Rift Valley, should be aware of the risk of contracting WNV and reduce exposure to mosquito bites, using insect repellents, and wearing protective clothing. The Ministry of Environmental Protection should be active in reducing the mosquito population by eliminating sources of standing water, a breeding ground for mosquitoes.

Finding a Needle in a Haystack: the Advantages of Liquid Chromatography--Tandem Mass Spectrometry (LC-MS/MS) in Determination of Sex Hormones in Children.

Determination of steroid sex hormones concentrations in children is very important for diagnosis of a wide range of pubertal, adrenal and sex development disorders. The majority of hormone measurements are carried out using traditional immunoassays, due to their technical simplicity, cost and availability of commercial reagents. But, due to limited specificity and sensitivity, traditional immunoassays often fail to determine low concentration analytes such as sex hormones in pediatric blood. In the last decade, the LC-MS/MS assay has risen as a new player in the analytic diagnostic field. The assay has proven appropriate for detection of very low hormones concentrations in blood, is quite easy to perform and can detect multiple steroids from a single sample. For the routine determination of an individual or panel of steroids, LC-MS/MS is now the recommended method for most diagnostic laboratories.

Is serum CRP level a reliable inflammatory marker in pediatric nephrotic syndrome?

BACKGROUND: This study tested the hypothesis that during massive proteinuria, C-reactive protein (CRP) may be lost into the urine along with other proteins, making serum CRP (sCRP) level an unreliable marker of infection severity in nephrotic syndrome (NS). METHODS: Children with active NS (n = 23) were compared with two matched control groups: patients with febrile non-renal infectious disease (n = 30) and healthy subjects (n = 16). Laboratory measurements included sCRP, urine protein, creatinine, IgG, and protein electrophoresis. Urinary CRP (uCRP) was measured by ELISA. RESULTS: Sixty-nine patients were enrolled: 23 patients with NS, 30 patients with non-renal febrile infectious diseases, and 16 healthy children. Median uCRP concentrations were 0 mcg/gCr (0-189.7) in NS, 11 mcg/gCr (0-286) in the febrile group, and 0 mcg/gCr (0-1.8) in the healthy group. The uCRP/creatinine ratio was similar in the NS and healthy groups (p > 0.1) and significantly higher in the febrile group than the other two groups (p < 0.0001). There was no association of uCRP concentration with severity of proteinuria or IgG excretion. CONCLUSIONS: NS in children is not characterized by significant loss of CRP into the urine. Therefore, sCRP may serve as a reliable marker of inflammation in this setting. The significant urinary excretion of CRP in children with transient non-renal infectious disease might be attributable to CRP synthesis in renal epithelial cells.

Structure activity relationship of carotenoid derivatives in activation of the electrophile/antioxidant response element transcription system.

Induction of phase II detoxifying enzymes is a major cellular strategy for reducing the risk of cancer. We previously reported that carotenoids activate the electrophile/antioxidant response element (EpRE/ARE) transcription system and induced the expression of phase II enzymes. Various electrophilic phytonutrients have been shown to induce the EpRE/ARE system by disrupting the inhibitory activity of Keap1 on Nrf2, the major EpRE/ARE activating transcription factor. However, hydrophobic carotenoids such as lycopene lack any electrophilic group and, thus, are unlikely to directly activate Nrf2 and the EpRE/ARE system. Here we demonstrate that carotenoid oxidation products are the active mediators in the stimulation of the EpRE/ARE system by carotenoids. Two lines of evidence support this conclusion. (A) The oxidized derivatives, extracted by ethanol from partially oxidized lycopene, transactivated EpRE/ARE with a potency similar to that of the unextracted lycopene mixture, whereas the intact carotenoid showed a nonsignificant effect. (B) Using a series of characterized mono- and diapocarotenoids that potentially can be derived from in vivo metabolism of carotenoids we defined the following structure-activity rules for activation of EpRE/ARE: (I) aldehydes and not acids are the active molecules; (II) the activity depends on the relative position of the methyl group to the terminal aldehyde which determines the reactivity of the conjugated double bond; (III) the optimal length of a dialdehyde derivative is 12 carbons in the main chain of the molecule. The apocarotenals inhibited breast and prostate cancer cell growth with a similar order of potency to the activation of EpRE/ARE. These results may provide a mechanistic explanation for the cancer preventive activity of carotenoids.

Carotenoids activate the antioxidant response element transcription system.

Epidemiologic studies have found an inverse association between consumption of tomato products and the risk of certain types of cancers. However, the mechanisms underlying this relationship are not completely understood. One mechanism that has been suggested is induction of phase II detoxification enzymes. Expression of phase II enzymes is regulated by the antioxidant response element (ARE) and the transcription factor Nrf2 (nuclear factor E2-related factor 2). In this study, we determined the role of this transcription system in the induction of phase II enzymes by carotenoids. We found that in transiently transfected cancer cells, lycopene transactivated the expression of reporter genes fused with ARE sequences. Other carotenoids such as phytoene, phytofluene, beta-carotene, and astaxanthin had a much smaller effect. An increase in protein as well as mRNA levels of the phase II enzymes NAD(P)H:quinone oxidoreductase and gamma-glutamylcysteine synthetase was observed in nontransfected cells after carotenoid treatment. Ethanolic extract of lycopene containing unidentified hydrophilic derivatives of the carotenoid activated ARE with similar potency to lycopene. The potency of the carotenoids in ARE activation did not correlate with their effect on intracellular reactive oxygen species and reduced glutathione level, which may indicate that ARE activation is not solely related to their antioxidant activity. Nrf2, which is found predominantly in the cytoplasm of control cells, translocated to the nucleus after carotenoid treatment. Interestingly, part of the translocated Nrf2 colocalized with the promyelocytic leukemia protein in the promyelocytic leukemia nuclear bodies. The increase in phase II enzymes was abolished by a dominant-negative Nrf2, suggesting that carotenoid induction of these proteins depends on a functional Nrf2 and the ARE transcription system.

Carotenoids and transcription.

The possible involvement of several transcription systems in the anticancer activity of carotenoids is the focus of this review. Carotenoids modulate the basic mechanisms of cell proliferation, growth factor signaling, gap junctional intercellular communication, and produce changes in the expression of many proteins participating in these processes. The changes in the expression of multiple proteins suggest that the initial effect of carotenoids involves modulation of transcription. We and others have found evidence for the role of several transcription systems, such as the retinoid receptors, activator protein-1 (AP-1), peroxisome proliferator-activated receptors (PPAR), xenobiotic receptors and the antioxidant response element (ARE), in the anticancer activity of carotenoids. The observed modulation of a network of transcription systems may provide the molecular basis for the synergistic anticancer effects of the combinations of various carotenoids together with other dietary and pharmacologic compounds.

Modulation of transcriptional activity by antioxidant carotenoids.

It is widely accepted that diet changes are a powerful means to prevent cancer. The possible involvement of transcriptional activity in the anticancer activity of carotenoids will be the focus of this review. Carotenoids function as potent antioxidants, and this is clearly a major mechanism of their action. In addition carotenoids action involves interference in several pathways related to cancer cell proliferation and includes changes in the expression of many proteins participating in these processes such as connexins, phase II enzymes, cyclins, cyclin-dependent kinases and their inhibitors. These changes in protein expression suggest that the initial effect involves modulation of transcription by ligand-activated nuclear receptors or by other transcription factors. It is feasible to suggest that carotenoids and their oxidized derivatives interact with a network of transcription systems that are activated by different ligands at low affinity and specificity and that this activation leads to the synergistic inhibition of cell growth.