Evaluation of sample pooling using the SAMBA II SARS-CoV-2 test.

BACKGROUND: Screening of infectious asymptomatic or pre-symptomatic individuals for SARS-CoV-2 is at present a key to controling the COVID-19 pandemic. In order to expand testing capability and limit cost, pool testing of asymtomatic individuals has been proposed, provided assay performance is not significantly affected. METHODS: Combined nose and throat (N/T) swabs collected from COVID-19 infected or non-infected individuals were tested using SAMBA II individually and in pools of four (one positive and 3 negative). The evaluation was conducted by the manufacturer and an independent NHS site. Ct cycles of individual positives and pooled positives were determined by qRT-PCR. RESULTS: In 42 pools containing a single positive sample with Ct values ranging between 17 and 36, 41 pools (97.6 %) were found positive by the SARS-CoV-2 SAMBA II test. The false-negative pool by SAMBA was also negative by both reference methods used in this evaluation.The individual positive sample in this pool was positive by SAMBA (Orf only) and by one of the reference methods (S gene only, Ct 35) but negative by the second reference method indicating that the sample itself was very low viral load. All 78 pools containing 4 negative swabs were negative (100 % specificity). DISCUSSION: The preliminary data of the evaluation indicated a high level of performance in both sensitivity and specificity of the SAMBA II assay when used to test pools of 4 patient samples. The implementation of this pooled protocol can increase throughput and reduce cost/test when the prevalence of COVID is low.

Single-shock defibrillation success in adult cardiac arrest: a systematic review.

OBJECTIVE: Current resuscitation guidelines advise a single biphasic shock followed by chest compressions; however, it is unclear if this applies to all waveforms and energy levels. We conducted a systematic review of the literature to determine the comparative success rates for single-shock defibrillation across waveforms evaluated in out-of-hospital cardiac arrest patients. METHODS: EMBASE, MEDLINE, EBM Reviews, dissertation abstract databases, and clinicaltrials.gov were searched. Two investigators independently reviewed titles, abstracts and full texts in a hierarchical manner for study eligibility with a quadratic kappa score at each level. Two authors abstracted data independently and the quality of the articles was assessed using the five-point Jadad scale. Outcomes were termination of ventricular fibrillation (VF)/ventricular tachycardia (VT) at 5s post shock (TOF), return of organized rhythm (ROOR) and return of spontaneous circulation (ROSC). RESULTS: A total of 3281 potentially relevant citations were identified and, of these, eight papers were selected with Kappa values of 0.53 for titles, 0.71 for abstracts, and 0.94 for articles. Quality scores varied from 0 to 4/5. Biphasic first-shock success for all three outcomes of interest was similar regardless of energy levels, and uniformly superior to monophasic first-shock success. Median time to first shock varied across trials based on level of randomization (first responders versus advanced life support tiered response) and may contribute to observed differences. Lack of variability across two waveforms precluded a meta-analytical approach. CONCLUSIONS: This systematic review suggests that evaluated biphasic waveforms have similar first-shock success as measured by the three outcomes of interest and all are superior to monophasic shocks.

Molecular basis of proton uptake in single and double mutants of cytochrome c oxidase.

Cytochrome c oxidase, the terminal enzyme of the respiratory chain, utilizes the reduction of dioxygen into water to pump protons across the mitochondrial inner membrane. The principal pathway of proton uptake into the enzyme, the D channel, is a 2.5 nm long channel-like cavity named after a conserved, negatively charged aspartic acid (D) residue thought to help recruiting protons to its entrance (D132 in the first subunit of the S. sphaeroides enzyme). The single-point mutation of D132 to asparagine (N), a neutral residue, abolishes enzyme activity. Conversely, replacing conserved N139, one-third into the D channel, by D, induces a decoupled phenotype, whereby oxygen reduction proceeds but not proton pumping. Intriguingly, the double mutant D132N/N139D, which conserves the charge of the D channel, restores the wild-type phenotype. We use molecular dynamics simulations and electrostatic calculations to examine the structural and physical basis for the coupling of proton pumping and oxygen chemistry in single and double N139D mutants. The potential of mean force for the conformational isomerization of N139 and N139D side chains reveals the presence of three rotamers, one of which faces the channel entrance. This out-facing conformer is metastable in the wild-type and in the N139D single mutant, but predominant in the double mutant thanks to the loss of electrostatic repulsion with the carboxylate group of D132. The effects of mutations and conformational isomerization on the pKa of E286, an essential proton-shuttling residue located at the top of the D channel, are shown to be consistent with the electrostatic control of proton pumping proposed recently (Fadda et al 2008 Biochim. Biophys. Acta 1777 277-84). Taken together, these results suggest that preserving the spatial distribution of charges at the entrance of the D channel is necessary to guarantee both the uptake and the relay of protons to the active site of the enzyme. These findings highlight the interplay of long-range electrostatic forces and local structural fluctuations in the control of proton movement and provide a physical explanation for the restoration of proton pumping activity in the double mutant.

Functional hydration and conformational gating of proton uptake in cytochrome c oxidase.

Cytochrome c oxidase couples the reduction of dioxygen to proton pumping against an electrochemical gradient. The D-channel, a 25-A-long cavity, provides the principal pathway for the uptake of chemical and pumped protons. A water chain is thought to mediate the relay of protons via a Grotthuss mechanism through the D-channel, but it is interrupted at N139 in all available crystallographic structures. We use free-energy simulations to examine the proton uptake pathway in the wild type and in single-point mutants N139V and N139A, in which redox and pumping activities are compromised. We present a general approach for the calculation of water occupancy in protein cavities and demonstrate that combining efficient sampling algorithms with long simulation times (hundreds of nanoseconds) is required to achieve statistical convergence of equilibrium properties in the protein interior. The relative population of different conformational and hydration states of the D-channel is characterized. Results shed light on the role of N139 in the mechanism of proton uptake and clarify the physical basis for inactive phenotypes. The conformational isomerization of the N139 side chain is shown to act as a gate controlling the formation of a functional water chain or "proton wire." In the closed state of N139, the spatial distribution of water in the D-channel is consistent with available crystallographic models. However, a metastable state of N139 opens up a narrow bottleneck in which 50% occupancy by a water molecule establishes a proton pathway throughout the D-channel. Results for N139V suggest that blockage of proton uptake resulting from persistent interruption of the water pathway is the cause of this mutant's marginal oxidase activity. In contrast, results for N139A indicate that the D-channel is a continuously hydrated cavity, implying that the decoupling of oxidase activity from proton pumping measured in this mutant is not due to interruption of the proton relay chain.