Use of Next-Generation Sequencing in a State-Wide Strategy of HIV-1 Surveillance: Impact of the SARS-COV-2 Pandemic on HIV-1 Diagnosis and Transmission.

BACKGROUND: The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic posed an unpreceded threat to the management of other pandemics such as human immunodeficiency virus-1 (HIV-1) in the United States. The full impact of the SARS-CoV-2 pandemic on the HIV-1 pandemic needs to be evaluated. METHODS: All individuals with newly reported HIV-1 diagnoses from NC State Laboratory of Public Health were enrolled in this prospective observational study, 2018-2021. We used a sequencing-based recency assay to identify recent HIV-1 infections and to determine the days postinfection (DPI) for each person at the time of diagnosis. RESULTS: Sequencing used diagnostic serum samples from 814 individuals with new HIV-1 diagnoses spanning this 4-year period. Characteristics of individuals diagnosed in 2020 differed from those in other years. People of color diagnosed in 2021 were on average 6 months delayed in their diagnosis compared to those diagnosed in 2020. There was a trend that genetic networks were more known for individuals diagnosed in 2021. We observed no major integrase resistance mutations over the course of the study. CONCLUSIONS: SARS-CoV-2 pandemic may contribute to the spread of HIV-1. Public health resources need to focus on restoring HIV-1 testing and interrupting active, ongoing, transmission.

RADx-UP Testing Core: Access to COVID-19 Diagnostics in Community-Engaged Research with Underserved Populations.

Research on the COVID-19 pandemic revealed a disproportionate burden of COVID-19 infection and death among underserved populations and exposed low rates of SARS-CoV-2 testing in these communities. A landmark National Institutes of Health (NIH) funding initiative, the Rapid Acceleration of Diagnostics-Underserved Populations (RADx-UP) program, was developed to address the research gap in understanding the adoption of COVID-19 testing in underserved populations. This program is the single largest investment in health disparities and community-engaged research in the history of the NIH. The RADx-UP Testing Core (TC) provides community-based investigators with essential scientific expertise and guidance on COVID-19 diagnostics. This commentary describes the first 2 years of the TC's experience, highlighting the challenges faced and insights gained to safely and effectively deploy large-scale diagnostics for community-initiated research in underserved populations during a pandemic. The success of RADx-UP shows that community-based research to increase access and uptake of testing among underserved populations can be accomplished during a pandemic with tools, resources, and multidisciplinary expertise provided by a centralized testing-specific coordinating center. We developed adaptive tools to support individual testing strategies and frameworks for these diverse studies and ensured continuous monitoring of testing strategies and use of study data. In a rapidly evolving setting of tremendous uncertainty, the TC provided essential and real-time technical expertise to support safe, effective, and adaptive testing. The lessons learned go beyond this pandemic and can serve as a framework for rapid deployment of testing in response to future crises, especially when populations are affected inequitably.

Efficient analytical gradients of property-based diabatic states: Geometry optimizations for localized holes.

We present efficient analytical gradients of property-based diabatic states and couplings using a Lagrangian formalism. Unlike previous formulations, the method achieves a computational scaling that is independent of the number of adiabatic states used to construct the diabats. The approach is generalizable to other property-based diabatization schemes and electronic structure methods as long as analytical energy gradients are available and integral derivatives with the property operator can be formed. We also introduce a scheme to phase and reorder diabats to ensure their continuity between molecular configurations. We demonstrate this for the specific case of Boys diabatic states obtained from state-averaged complete active space self-consistent field electronic structure calculations with GPU acceleration in the TeraChem package. The method is used to test the Condon approximation for the hole transfer in an explicitly solvated model DNA oligomer.

Evidence for Water Antibonding Orbital Mixing in the Hydrated Electron from Its Oxygen 1s X-ray Absorption Spectrum.

The X-ray absorption spectrum (XAS) of the hydrated electron (e(aq)-) has been simulated using time-dependent density functional theory with a quantum mechanics/molecular mechanics description. A unique XAS peak at 533 eV is observed with an energy and intensity in quantitative agreement with recent time-resolved experiments, allowing its assignment as arising from water O1s transitions to the singly occupied molecular orbital (SOMO) in which the excess electron resides. The transitions acquire oscillator strength due to the SOMO comprising an admixture of a cavity-localized orbital and water 4a1 and 2b2 antibonding orbitals. The mixing of antibonding orbitals has implications for the strength of couplings between e(aq)- and intramolecular modes of water.

Ultrafast Photoisomerization of Ethylene Studied Using Time-Resolved Extreme Ultraviolet Photoelectron Spectroscopy.

The photoisomerization of isolated ethylene (ethene) was observed in real time from the Franck-Condon region in the 1ππ* state to ground-state products using time-resolved photoelectron spectroscopy with extreme ultraviolet (EUV, 21.7 eV) probe pulses. A combination of filamentation four-wave mixing and high-order harmonic generation was employed to obtain a temporal resolution of 31 ± 2 fs. The nuclear wave packet created by a 160 nm pump pulse accesses C═C twisted geometries within 10 fs, and the population transfer from the excited to the ground state occurs within the next 20-30 fs. Formation of vibrationally highly excited ground-state molecules was observed in less than 45 fs, and they decayed with two time constants of 0.87 and >5 ps. The interpretation of the photoelectron spectra is supported by vertical ionization energies calculated using XMS-CASPT2 along geodesically interpolated reaction paths from the Franck-Condon region to the products.

Flexible boundary layer using exchange for embedding theories. I. Theory and implementation.

Embedding theory is a powerful computational chemistry approach to exploring the electronic structure and dynamics of complex systems, with Quantum Mechanical/Molecular Mechanics (QM/MM) being the prime example. A challenge arises when trying to apply embedding methodology to systems with diffusible particles, e.g., solvents, if some of them must be included in the QM region, for example, in the description of solvent-supported electronic states or reactions involving proton transfer or charge-transfer-to-solvent: without a special treatment, inter-diffusion of QM and MM particles will eventually lead to a loss of QM/MM separation. We have developed a new method called Flexible Boundary Layer using Exchange (FlexiBLE) that solves the problem by adding a biasing potential to the system that closely maintains QM/MM separation. The method rigorously preserves ensemble averages by leveraging their invariance to an exchange of identical particles. With a careful choice of the biasing potential and the use of a tree algorithm to include only important QM and MM exchanges, we find that the method has an MM-forcefield-like computational cost and thus adds negligible overhead to a QM/MM simulation. Furthermore, we show that molecular dynamics with the FlexiBLE bias conserves total energy, and remarkably, sub-diffusional dynamical quantities in the inner QM region are unaffected by the applied bias. FlexiBLE thus widens the range of chemistry that can be studied with embedding theory.

Flexible boundary layer using exchange for embedding theories. II. QM/MM dynamics of the hydrated electron.

The FlexiBLE embedding method introduced in Paper I [Z. Shen and W. J. Glover, J. Chem. Phys. 155, 224112 (2021)] is applied to explore the structure and dynamics of the aqueous solvated electron at an all-electron density functional theory Quantum Mechanics/Molecular Mechanics level. Compared to a one-electron mixed quantum/classical description, we find the dynamics of the many-electron model of the hydrated electron exhibits enhanced coupling to water OH stretch modes. Natural bond orbital analysis reveals this coupling is due to significant population of water OH σ* orbitals, reaching 20%. Based on this, we develop a minimal frontier orbital picture of the hydrated electron involving a cavity orbital and important coupling to 4-5 coordinating OH σ* orbitals. Implications for the interpretation of the spectroscopy of this interesting species are discussed.

Active orbital preservation for multiconfigurational self-consistent field.

We introduce Active Orbital Preservation for Multiconfigurational Self-Consistent Field (AOP-MCSCF), an automated approach to improving the consistency of active space orbitals over multiple molecular configurations. Our approach is based on maximum overlap with a reference set of active space orbitals taken from a single geometry of a chromophore in the gas phase and can be used to automatically preserve the appropriate orbitals of the chromophore across multiple thermally sampled configurations, even when the chromophore is solvated by quantum-mechanically treated water molecules. In particular, using the singular value decomposition of a Molecular Orbital (MO) overlap matrix between the system and reference, we rotate the MOs of the system to align with the reference active space orbitals and use the resulting rotated orbitals as an initial guess to a MCSCF calculation. We demonstrate the approach on aqueous p-hydroxybenzylidene-imidazolinone (HBI) and find that AOP-MCSCF converges to the "correct" orbitals for over 90% of 3000 thermally sampled configurations. In addition, we compute the linear absorption spectrum and find excellent agreement with new experimental measurements up to 5.4 eV (230 nm). We show that electrostatic contributions to the solvation energy of HBI largely explain the observed state-dependent solvatochromism.

Accurate Prediction of Absorption Spectral Shifts of Proteorhodopsin Using a Fragment-Based Quantum Mechanical Method.

Many experiments have been carried out to display different colors of Proteorhodopsin (PR) and its mutants, but the mechanism of color tuning of PR was not fully elucidated. In this study, we applied the Electrostatically Embedded Generalized Molecular Fractionation with Conjugate Caps (EE-GMFCC) method to the prediction of excitation energies of PRs. Excitation energies of 10 variants of Blue Proteorhodopsin (BPR-PR105Q) in residue 105GLN were calculated with the EE-GMFCC method at the TD-B3LYP/6-31G* level. The calculated results show good correlation with the experimental values of absorption wavelengths, although the experimental wavelength range among these systems is less than 50 nm. The ensemble-averaged electric fields along the polyene chain of retinal correlated well with EE-GMFCC calculated excitation energies for these 10 PRs, suggesting that electrostatic interactions from nearby residues are responsible for the color tuning. We also utilized the GMFCC method to decompose the excitation energy contribution per residue surrounding the chromophore. Our results show that residues ASP97 and ASP227 have the largest contribution to the absorption spectral shift of PR among the nearby residues of retinal. This work demonstrates that the EE-GMFCC method can be applied to accurately predict the absorption spectral shifts for biomacromolecules.

SSAIMS-Stochastic-Selection Ab Initio Multiple Spawning for Efficient Nonadiabatic Molecular Dynamics.

Ab initio multiple spawning provides a powerful and accurate way of describing the excited-state dynamics of molecular systems, whose strength resides in the proper description of coherence effects during nonadiabatic processes thanks to the coupling of trajectory basis functions. However, the simultaneous propagation of a large number of trajectory basis functions can be numerically inconvenient. We propose here an elegant and simple solution to this issue, which consists of (i) detecting uncoupled groups of coupled trajectory basis functions and (ii) selecting stochastically one of these groups to continue the ab initio multiple spawning dynamics. We show that this procedure can reproduce the results of full ab initio multiple spawning dynamics in cases where the uncoupled groups of trajectory basis functions stay uncoupled throughout the dynamics (which is often the case in high-dimensional problems). We present and discuss the aforementioned idea in detail and provide simple numerical applications on indole, ethylene, and protonated formaldimine, highlighting the potential of stochastic-selection ab initio multiple spawning.

Prediction of Excited-State Properties of Oligoacene Crystals Using Fragment-Based Quantum Mechanical Method.

A fundamental understanding of the excited-state properties of molecular crystals is of central importance for their optoelectronics applications. In this study, we developed the electrostatically embedded generalized molecular fractionation (EE-GMF) method for the quantitative characterization of the excited-state properties of locally excited molecular clusters. The accuracy of the EE-GMF method is systematically assessed for oligoacene crystals. Our result demonstrates that the EE-GMF method is capable of providing the lowest vertical singlet (S1) and triplet excitation energies (T1), in excellent agreement with the full-system quantum mechanical calculations. Using this method, we also investigated the performance of different density functionals in predicting the excited-state properties of the oligoacene crystals. Among the 13 tested functionals, B3LYP and MN15 give the two lowest overall mean unsigned errors with reference to the experimental S1 and T1 excitation energies. The EE-GMF approach can be readily utilized for studying the excited-state properties of large-scale organic solids at diverse ab initio levels.

Psychrobacter sanguinis Wound Infection Associated with Marine Environment Exposure, Washington, USA.

We report a 26-year-old man with Psychrobacter sanguinis cellulitis of a wound sustained during ocean fishing in Washington, USA, in 2017. Psychrobacter spp. are opportunistic pathogens found in a wide range of environments. Clinicians should be aware of Psychrobacter spp. and perform 16S rRNA sequencing if this pathogen is suspected.

Fly Reservoir Associated with Wohlfahrtiimonas Bacteremia in a Human.

Wohlfahrtiimonas species bacteria were isolated from the bloodstream of a patient with septicemia and wound myiasis. Environmental investigations identified a Wohlfahrtiimonas sp. among insects in the Americas and in a previously undescribed vector, the green bottle fly (Lucilia sericata). The isolates possibly represent a new species within the genus Wohlfahrtiimonas.

Excited state non-adiabatic dynamics of the smallest polyene, trans 1,3-butadiene. II. Ab initio multiple spawning simulations.

The excited state non-adiabatic dynamics of the smallest polyene, trans 1,3-butadiene (BD), has long been the subject of controversy due to its strong coupling, ultrafast time scales and the difficulties that theory faces in describing the relevant electronic states in a balanced fashion. Here we apply Ab Initio Multiple Spawning (AIMS) using state-averaged complete active space multistate second order perturbation theory [SA-3-CAS(4/4)-MSPT2] which describes both static and dynamic electron correlation effects, providing a balanced description of both the initially prepared bright 11Bu (ππ*) state and non-adiabatically coupled dark 21Ag state of BD. Importantly, AIMS allows for on-the-fly calculations of experimental observables. We validate our approach by directly simulating the time resolved photoelectron-photoion coincidence spectroscopy results presented in Paper I [A. E. Boguslavskiy et al., J. Chem. Phys. 148, 164302 (2018)], demonstrating excellent agreement with experiment. Our simulations reveal that the initial excitation to the 11Bu state rapidly evolves via wavepacket dynamics that follow both bright- and dark-state pathways as well as mixtures of these. In order to test the sensitivity of the AIMS results to the relative ordering of states, we considered two hypothetical scenarios biased toward either the bright 1Bu or the dark 21Ag state. In contrast with AIMS/SA-3-CAS(4/4)-MSPT2 simulations, neither of these scenarios yields favorable agreement with experiment. Thus, we conclude that the excited state non-adiabatic dynamics in BD involves both of these ultrafast pathways.

Excited state non-adiabatic dynamics of the smallest polyene, trans 1,3-butadiene. I. Time-resolved photoelectron-photoion coincidence spectroscopy.

The ultrafast excited state dynamics of the smallest polyene, trans-1,3-butadiene, were studied by femtosecond time-resolved photoelectron-photoion coincidence (TRPEPICO) spectroscopy. The evolution of the excited state wavepacket, created by pumping the bright 1Bu (ππ*) electronic state at its origin of 216 nm, is projected via one- and two-photon ionization at 267 nm onto several ionization continua. The results are interpreted in terms of Koopmans' correlations and Franck-Condon factors for the excited and cationic states involved. The known predissociative character of the cation excited states is utilized to assign photoelectron bands to specific continua using TRPEPICO spectroscopy. This permits us to report the direct observation of the famously elusive S1(21Ag) dark electronic state during the internal conversion of trans 1,3-butadiene. Our phenomenological analysis permits the spectroscopic determination of several important time constants. We report the overall decay lifetimes of the 11Bu and 21Ag states and observe the re-appearance of the hot ground state molecule. We argue that the apparent dephasing time of the S2(11Bu) state, which leads to the extreme breadth of the absorption spectrum, is principally due to large amplitude torsional motion on the 1Bu surface in conjunction with strong non-adiabatic couplings via conical intersections, whereupon nuclear wavepacket revivals to the initial Franck-Condon region become effectively impossible. In Paper II [W. J. Glover et al., J. Chem. Phys. 148, 164303 (2018)], ab initio multiple spawning is used for on-the-fly computations of the excited state non-adiabatic wavepacket dynamics and their associated TRPEPICO observables, allowing for direct comparisons of experiment with theory.

Whole genome sequencing analyses of Listeria monocytogenes that persisted in a milkshake machine for a year and caused illnesses in Washington State.

BACKGROUND: In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes. RESULTS: The outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA. Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the AscI restriction site in the prophage, which contributed to their AscI-PFGE banding pattern differences from other isolates. CONCLUSIONS: The high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

Legionnaires' disease in transplant recipients: A 15-year retrospective study in a tertiary referral center.

Legionnaires' disease (LD) can be fatal among high-risk transplant recipients. To understand the epidemiology of LD, we reviewed 15-year longitudinal data from a center in Seattle, Washington that cares for both solid organ transplant (SOT) and hematopoietic cell transplant (HCT) recipients. We identified all laboratory-confirmed LD and extracted data on species, diagnostic modalities, clinical presentation, management, and outcomes from medical records. Among 32 patients with LD, transplant recipients made up the majority of diagnoses (22, 69%; SOT 10, HCT 12). Approximately 0.8% of transplant recipients who underwent Legionella-specific testing were positive. Non-pneumophila Legionella species (LNLP), which are not detected by urinary antigen test, accounted for half the cases, led by Legionella micdadei (32%). The severity and outcome between Legionella pneumophila and LNLP infections were similar (attributed mortality, 36% vs 27%); all LNLP deaths occurred in transplant recipients with L. micdadei. The clinical and radiological features mimicked other opportunistic pathogens; 32% (n=7) were not on empiric treatment at the time of diagnosis. These data add to the emerging literature describing the importance of LD and highlight the need for both routine Legionella testing on transplant recipients with pulmonary findings and empiric Legionella-active antibiotic therapy.

Temperature dependence of the hydrated electron's excited-state relaxation. I. Simulation predictions of resonance Raman and pump-probe transient absorption spectra of cavity and non-cavity models.

We use one-electron non-adiabatic mixed quantum/classical simulations to explore the temperature dependence of both the ground-state structure and the excited-state relaxation dynamics of the hydrated electron. We compare the results for both the traditional cavity picture and a more recent non-cavity model of the hydrated electron and make definite predictions for distinguishing between the different possible structural models in future experiments. We find that the traditional cavity model shows no temperature-dependent change in structure at constant density, leading to a predicted resonance Raman spectrum that is essentially temperature-independent. In contrast, the non-cavity model predicts a blue-shift in the hydrated electron's resonance Raman O-H stretch with increasing temperature. The lack of a temperature-dependent ground-state structural change of the cavity model also leads to a prediction of little change with temperature of both the excited-state lifetime and hot ground-state cooling time of the hydrated electron following photoexcitation. This is in sharp contrast to the predictions of the non-cavity model, where both the excited-state lifetime and hot ground-state cooling time are expected to decrease significantly with increasing temperature. These simulation-based predictions should be directly testable by the results of future time-resolved photoelectron spectroscopy experiments. Finally, the temperature-dependent differences in predicted excited-state lifetime and hot ground-state cooling time of the two models also lead to different predicted pump-probe transient absorption spectroscopy of the hydrated electron as a function of temperature. We perform such experiments and describe them in Paper II [E. P. Farr et al., J. Chem. Phys. 147, 074504 (2017)], and find changes in the excited-state lifetime and hot ground-state cooling time with temperature that match well with the predictions of the non-cavity model. In particular, the experiments reveal stimulated emission from the excited state with an amplitude and lifetime that decreases with increasing temperature, a result in contrast to the lack of stimulated emission predicted by the cavity model but in good agreement with the non-cavity model. Overall, until ab initio calculations describing the non-adiabatic excited-state dynamics of an excess electron with hundreds of water molecules at a variety of temperatures become computationally feasible, the simulations presented here provide a definitive route for connecting the predictions of cavity and non-cavity models of the hydrated electron with future experiments.

Two Listeria monocytogenes Pseudo-outbreaks Caused by Contaminated Laboratory Culture Media.

Listeriosis is a serious foodborne infection that disproportionately affects elderly adults, pregnant women, newborns, and immunocompromised individuals. Diagnosis is made by culturing Listeria monocytogenes from sterile body fluids or from products of conception. This report describes the investigations of two listeriosis pseudo-outbreaks caused by contaminated laboratory media made from sheep blood.

Evaluation and utilization of preassembled frozen commercial fast real-time qPCR master mixes for detection of cytomegalovirus and BK virus.

Quantitative DNA detection of cytomegalovirus (CMV) and BK virus (BKV) is critical in the management of transplant patients. Quantitative laboratory-developed procedures for CMV and BKV have been described in which much of the processing is automated, resulting in rapid, reproducible, and high-throughput testing of transplant patients. To increase the efficiency of such assays, the performance and stability of four commercial preassembled frozen fast qPCR master mixes (Roche FastStart Universal Probe Master Mix with Rox, Bio-Rad SsoFast Probes Supermix with Rox, Life Technologies TaqMan FastAdvanced Master Mix, and Life Technologies Fast Universal PCR Master Mix), in combination with in-house designed primers and probes, was evaluated using controls and standards from standard CMV and BK assays. A subsequent parallel evaluation using patient samples was performed comparing the performance of freshly prepared assay mixes versus aliquoted frozen master mixes made with two of the fast qPCR mixes (Life Technologies TaqMan FastAdvanced Master Mix, and Bio-Rad SsoFast Probes Supermix with Rox), chosen based on their performance and compatibility with existing PCR cycling conditions. The data demonstrate that the frozen master mixes retain excellent performance over a period of at least 10 weeks. During the parallel testing using clinical specimens, no difference in quantitative results was observed between the preassembled frozen master mixes and freshly prepared master mixes. Preassembled fast real-time qPCR frozen master mixes perform well and represent an additional strategy laboratories can implement to reduce assay preparation times, and to minimize technical errors and effort necessary to perform clinical PCR.