Implementation of a Sample Pooling Strategy for the Direct Detection of SARS-CoV-2 by Real-Time Polymerase Chain Reaction During the COVID-19 Pandemic.

OBJECTIVES: To report our institutional experience in devising and implementing a pooling protocol and process for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) reverse transcription polymerase chain reaction (RT-PCR) testing over a 3-month period in the fall of 2020. METHODS: The widespread testing implemented in the United States for detecting SARS-CoV-2 infection in response to the coronavirus disease 2019 pandemic has led to a significant shortage of testing supplies and therefore has become a major impediment to the public health response. To date, several institutions have implemented sample pooling, but publications documenting these experiences are sparse. Nasal and nasopharyngeal samples collected from low-positivity (<5%) areas were tested in pools of five on the Roche cobas 6800 analyzer system. Routine SARS-CoV-2 RT-PCR turnaround times between sample collection to result reporting were monitored and compared before and after sample pooling implementation. RESULTS: A total of 4,131 sample pools were tested over a 3-month period (during which 39,770 RT-PCR results were reported from the Roche system), allowing our laboratory to save 13,824 tests, equivalent to a conservation rate of 35%. A 48-hour or less turnaround time was generally maintained throughout the pooling period. CONCLUSIONS: Sample pooling offers a viable means to mitigate shortfalls of PCR testing supplies in the ongoing pandemic without significantly compromising overall turnaround times.

Cytoreduction with hyperthermic intra peritoneal and intra thoracic chemotherapy for metastatic Sertoli-Leydig cell tumor of the ovary.

Sertoli Leydig cell tumor (SLCT) is a rare sex-cord stromal tumor of the ovary that generally has a benign course. Here, we report an unusual case of recurrent, metastatic SLCT and its unique management with a combination of cytoreductive surgery, hyperthermic intraperitoneal chemotherapy, hyperthermic intrathoracic chemotherapy, and systemic chemotherapy.

Effect of antimicrobial stewardship with rapid MALDI-TOF identification and Vitek 2 antimicrobial susceptibility testing on hospitalization outcome.

We sought to evaluate time for bacterial culture and initiation of appropriate therapy for patients, from 2017 (without rapid diagnostic test (RDT) and Antimicrobial Stewardship Program (ASP)) and 2018 (with RDT and ASP). Time to identification (ID) was significantly faster in 2018 (2018 24.9 ±â€¯14.4, 2017 33.8 ±â€¯17 h, P = 0.001). Time to antimicrobial susceptibility test (AST) results was significantly faster for patients in 2018 compared to 2017 (18.2 ±â€¯14 h compared to 28.5 ±â€¯14.9 h, P < 0.001). Length of hospital stay for enrolled patients was significantly shorter in 2018 compared to 2017 (2018 10.7 ±â€¯11.1 days and 2017 15.5 ±â€¯18.1 days, P = 0.05). Length of antimicrobial therapy for enrolled patients was significantly shorter for 2018 (2018 6.7 ±â€¯3.8 d vs. 2017 8.8 ±â€¯7.8 d, P = 0.036). These results demonstrate MALDI-TOF/Vitek 2 leads to an average 21.5 h faster ID and AST results that can be acted upon by ASP for antimicrobial recommendations.

Leukocyte filtration and postoperative infections.

BACKGROUND: Leukocyte filtration has been hypothesized to reduce the risk of postoperative infections by alleviating the immunosuppressive effect of whole blood. However, the literature regarding the clinical efficacy of leukocyte filtration remains conflicted. This meta-analysis investigates the impact of allogeneic and autologous leukocyte-filtered blood transfusions on the incidence of postoperative infections in adult surgical patients. METHODS: A comprehensive literature search of PubMed, Google Scholar, and Cochrane Central Registry of Controlled trials (1966-2016) was completed for all published randomized controlled trials. Postoperative infections under "as-per-protocol" (APP) and "intention-to-treat" (ITT), length of stay, and mortality were analyzed. RESULTS: Sixteen randomized controlled trials involving 6586 randomized (ITT) patients (4615 APP patients) in various clinical settings were evaluated. The leukocyte-filtered blood group demonstrated an overall 26% risk reduction in postoperative infections when analyzed by APP (relative risk [RR] = 0.74; 95% confidence interval [CI, 0.60-0.92]; P = 0.007) and a 22% risk reduction when analyzed by ITT (RR = 0.78; 95% CI [0.65-0.94]; P = 0.009). Leukocyte-filtered blood was also associated with a significant reduction in length of stay (standardized difference of mean [SDM] = -0.74; 95% CI [-1.32 to -0.15]; P = 0.014) and all-cause mortality (RR = 0.74; 95% CI [0.57-0.95]; P = 0.018). CONCLUSIONS: Leukocyte-filtered blood transfusions are associated with significantly lower postoperative infection rates in both the APP and ITT populations. Leukocyte filtration also shortens length of stay and decreases all-cause mortality in surgical patients and should be considered in all surgical patients.

National Institute of Allergy and Infectious Disease (NIAID) Funding for Studies of Hospital-Associated Bacterial Pathogens: Are Funds Proportionate to Burden of Disease?

BACKGROUND: Hospital-associated infections (HAIs) are associated with a considerable burden of disease and direct costs greater than $17 billion. The pathogens that cause the majority of serious HAIs are Enterococcus faecium, Staphylococcus aureus, Clostridium difficile, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species, referred as ESCKAPE. We aimed to determine the amount of funding the National Institute of Health (NIH) National Institute of Allergy and Infectious Diseases (NIAID) allocates to research on antimicrobial resistant pathogens, particularly ESCKAPE pathogens. METHODS: The NIH Research Portfolio Online Reporting Tools (RePORT) database was used to identify NIAID antimicrobial resistance research grants funded in 2007-2009 using the terms "antibiotic resistance," "antimicrobial resistance," and "hospital-associated infection." RESULTS: Funding for antimicrobial resistance grants has increased from 2007-2009. Antimicrobial resistance funding for bacterial pathogens has seen a smaller increase than non-bacterial pathogens. The total funding for all ESKCAPE pathogens was $ 22,005,943 in 2007, $ 30,810,153 in 2008 and $ 49,801,227 in 2009. S. aureus grants received $ 29,193,264 in FY2009, the highest funding amount of all the ESCKAPE pathogens. Based on 2009 funding data, approximately $1,565 of research money was spent per S. aureus related death and $750 of was spent per C. difficile related death. CONCLUSIONS: Although the funding for ESCKAPE pathogens has increased from 2007 to 2009, funding levels for antimicrobial resistant bacteria-related grants is still lower than funding for antimicrobial resistant non-bacterial pathogens. Efforts may be needed to improve research funding for resistant-bacterial pathogens, particularly as their clinical burden increases.

Crystal structure of lactose permease in complex with an affinity inactivator yields unique insight into sugar recognition.

Lactose permease of Escherichia coli (LacY) with a single-Cys residue in place of A122 (helix IV) transports galactopyranosides and is specifically inactivated by methanethiosulfonyl-galactopyranosides (MTS-gal), which behave as unique suicide substrates. In order to study the mechanism of inactivation more precisely, we solved the structure of single-Cys122 LacY in complex with covalently bound MTS-gal. This structure exhibits an inward-facing conformation similar to that observed previously with a slight narrowing of the cytoplasmic cavity. MTS-gal is bound covalently, forming a disulfide bond with C122 and positioned between R144 and W151. E269, a residue essential for binding, coordinates the C-4 hydroxyl of the galactopyranoside moiety. The location of the sugar is in accord with many biochemical studies.

Bridging the gap: a GFP-based strategy for overexpression and purification of membrane proteins with intra and extracellular C-termini.

Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C(in)) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C(out)) to C(in) ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C(out) topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescence-detection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C(out) topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.

Fluorescence Detection of Heavy Atom Labeling (FD-HAL): a rapid method for identifying covalently modified cysteine residues by phasing atoms.

Membrane protein crystallography frequently stalls at the phase determination stage due to poor crystal diffraction and the inability to identify heavy atom derivatization prior to data collection. Thus, a majority of time, effort and resources are invested preparing potential derivatized crystals for synchrotron data collection and analysis without knowledge of heavy atom labeling. To remove this uncertainty, we introduce Fluorescence Detection of Heavy Atom Labeling (FD-HAL) using tetramethylrhodamine-5-maleimide (a fluorescent maleimide compound) to monitor in-gel cysteine residue accessibility and ascertain covalent modification by mercury, platinum and gold compounds. We have tested this technique on three integral membrane proteins (LacY, vSGLT and mVDAC1) and can quickly assess the optimal concentrations, time and heavy atom compound to derivatize free cysteine residues in order to facilitate crystal phasing. This, in conjunction with cysteine scanning for incorporating heavy atoms at strategic positions, is a useful tool that will considerably assist in phasing membrane protein structures.

The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+ binding motif.

The Na+/Ca2+ exchanger is a plasma membrane protein that regulates intracellular Ca2+ levels in cardiac myocytes. Transport activity is governed by Ca2+, and the primary Ca2+ sensor (CBD1) is located in a large cytoplasmic loop connecting two transmembrane helices. The binding of Ca2+ to the CBD1 sensory domain results in conformational changes that stimulate the exchanger to extrude Ca2+. Here, we present a crystal structure of CBD1 at 2.5A resolution, which reveals a novel Ca2+ binding site consisting of four Ca2+ ions arranged in a tight planar cluster. This intricate coordination pattern for a Ca2+ binding cluster is indicative of a highly sensitive Ca2+ sensor and may represent a general platform for Ca2+ sensing.