Molecular Identification of Fonsecaea monophora, Novel Agent of Fungal Brain Abscess.

A 3-year-old patient in India experiencing headaches and seizures was diagnosed with a fungal infection, initially misidentified as Cladophialophora bantiana. Follow-up sequencing identified the isolate to be Fonsecaea monophora fungus. This case demonstrates the use of molecular methods for the correct identification of F. monophora, an agent of fungal brain abscess.

Utility of an in-house real-time PCR in whole blood samples as a minimally invasive method for early and accurate diagnosis of invasive mould infections.

INTRODUCTION: Invasive mould infections (IMIs) are a leading cause of death in patients with compromised immune systems. Proven invasive mould infection requires detection of a fungus by histopathological analysis of a biopsied specimen, sterile culture, or fungal DNA amplification by PCR in tissue. However, the clinical performance of a PCR assay on blood samples taken from patients suspected of invasive mould disease has not been fully evaluated, particularly for the differential diagnosis of invasive aspergillosis (IA) and invasive Mucormycosis (IM). OBJECTIVES: To assess the diagnostic utility of our previously validated in-house real-time PCR in blood samples for diagnosis of invasive aspergillosis and mucormycosis in patients with suspected invasive mould infection. METHODS: All patients with suspected invasive mould infection were prospectively enrolled from May 2021 to July 2021. Conventional fungal diagnosis was performed using tissue and respiratory samples. In-house PCR was performed on blood samples and its diagnostic performance evaluated. RESULTS: A total of 158 cases of suspected invasive mould infection were enrolled in the study. The sensitivity and specificity of in-house PCR performed on blood samples was found to be 92.5% and 81.4% respectively for diagnosis of probable IA, and 65% and 84.62% respectively for diagnosis of proven and probable IM. It was also able to detect 3 out of 5 cases of possible IM where no other microbiological evidence of IM was obtained. CONCLUSIONS: This assay could be helpful in minimally invasive diagnosis of IMIs for patients in whom invasive sampling is not feasible, especially as a preliminary or screening test. It can help in early diagnosis, anticipating conventional laboratory confirmation by days or weeks. Possible correlation between fungal load and mortality can help in initiating aggressive treatment for patients with high initial fungal load.

Effect of volume of instillate on the diagnostic utility of bronchoalveolar lavage galactomannan in patients with suspected chronic pulmonary aspergillosis-A pilot study.

BACKGROUND: Bronchoalveolar lavage (BAL) galactomannan (GM) is commonly used to diagnose Aspergillus-related lung diseases. However, unlike serum GM, which is measured in undiluted blood, BAL-GM is estimated using variable aliquots and cumulative volume of instillates during bronchoscopy. OBJECTIVE: Since different studies have reported varying diagnostic accuracy and cut-offs for BAL-GM in CPA, we hypothesized that the total volume of instillate and 'order/label' of aliquots significantly affects the BAL-GM values, which was evaluated as part of this study. PATIENTS & METHODS: We obtained 250 BAL samples from 50 patients (five from each) with suspected chronic pulmonary aspergillosis. BAL fluid was collected after instilling sequential volumes of 40 mL of normal saline each for the first four labels and a fifth label was prepared by mixing 1 mL from each of the previous labels. The GM level of each label was measured by PLATELIA™ ASPERGILLUS Ag enzyme immunoassay. This study measured the discordance, level of agreement, diagnostic characteristics (sensitivity, specificity and AUROC) and best cut-offs for BAL-GM in the different aliquots of lavage fluid. RESULTS: The study population, classified into CPA (28%) and non-CPA (72%) groups, based on ERS/ESCMID criteria (excluding BAL-GM) were not different with respect to clinico-radiological characteristics. The discordance of BAL-GM positivity (using a cut-off of >1) between the serial labels for the same patient ranged between 10% and 22%, while the discordance between classification using BAL-GM positivity (using a cut-off of ≥1) and clinic-radio-microbiological classification ranged between 18% and 30%. The level of agreement for serial labels was at best fair (<0.6 for all except one 'label'). The AUROC for the serial samples ranged between 0.595 and 0.702, with the '40 mL and the 'mix' samples performing the best. The best BAL-GM cut-off also showed significant variation between serial labels of varying dilutions (Range:1.01 - 4.26). INTERPRETATION: This study highlights the variation in BAL-GM measured and the 'positivity' between different 'labels' of aliquots of BAL, with the first aliquot and the mixed sample showing the best performances for diagnosis of CPA. Future studies should attempt to 'standardise' the instilled volume for BAL-GM estimation to standardise the diagnostic yield.

Simulating the Excited-State Dynamics of Polaritons with Ab Initio Multiple Spawning.

Over the past decade, there has been a growth of interest in polaritonic chemistry, where the formation of hybrid light-matter states (polaritons) can alter the course of photochemical reactions. These hybrid states are created by strong coupling between molecules and photons in resonant optical cavities and can even occur in the absence of light when the molecule is strongly coupled with the electromagnetic fluctuations of the vacuum field. We present a first-principles model to simulate nonadiabatic dynamics of such polaritonic states inside optical cavities by leveraging graphical processing units (GPUs). Our first implementation of this model is specialized for a single molecule coupled to a single-photon mode confined inside the optical cavity but with any number of excited states computed using complete active space configuration interaction (CASCI) and a Jaynes-Cummings-type Hamiltonian. Using this model, we have simulated the excited-state dynamics of a single salicylideneaniline (SA) molecule strongly coupled to a cavity photon with the ab initio multiple spawning (AIMS) method. We demonstrate how the branching ratios of the photodeactivation pathways for this molecule can be manipulated by coupling to the cavity. We also show how one can stop the photoreaction from happening inside of an optical cavity. Finally, we also investigate cavity-based control of the ordering of two excited states (one optically bright and the other optically dark) inside a cavity for a set of molecules, where the dark and bright states are close in energy.

Utility of in-house and commercial PCR assay in diagnosis of Covid-19 associated mucormycoss in an emergency setting in a tertiary care center.

Introduction. Invasive mucormycosis (IM) is a potentially fatal infection caused by fungi of the order Mucorales. Histopathology, culture, and radiology are the mainstays of diagnosis, but they are not sufficiently sensitive, resulting in delayed diagnosis and intervention. Recent studies have shown that PCR-based techniques can be a promising way to diagnose IM.Hypothesis/Gap Statement. Early diagnosis of fungal infections using molecular diagnostic techniques can improve patient outcomes, especially in invasive mucormycosis.Aim. The aim of this study was to evaluate the utility of our in-house mould-specific real time PCR assay (qPCR) in comparison with the commercially available real time PCR (MucorGenius PCR), for the early diagnosis of mucormycosis in tissue samples from patients with suspicion of invasive mucormycosis (IM). This in-house assay can detect and distinguish three clinically relevant mould species, e.g. Aspergillus spp., Mucorales and Fusarium spp. in a single reaction with only one pair of primers, without the need for sequencing.Methodology. We enrolled 313 tissue samples from 193 patients with suspected IM in this prospective study. All cases were classified using EORTC/MSGERC guidelines. All samples were tested using traditional methods, in-house qPCR, and MucorGenius PCR.Results. Using direct microscopy as a gold standard, the overall sensitivity and specificity of in-house qPCR for detection of IM was 92.46% and 80% respectively, while that of the MucorGenius PCR was 66.67% and 90% respectively. However, co-infection of IM and IA adversely affected the performance of MucorGenius PCR in detection of IM.The in-house PCR detected Aspergillus spp. in 14 cases and Fusarium spp. in 4 cases which showed clinical and radiological features of fungal sinusitis. The in-house qPCR also performed better in detecting possible cases of IM. This aids early diagnosis and appropriate treatment to improve patient outcomes.Conclusion. Because the in-house PCR is not only sensitive and specific, but also entirely based on SYBR Green for detection of targets, it is less expensive than probe-based assays and can be used on a regular basis for the diagnosis of IM in resource-constrained settings. It can be used to distinguish between mucormycosis and fungal sinusitis caused by Aspergillus and Fusarium in high-risk patients, as well as to accurately detect Mucorales in fungal co-infection cases.

A study on candiduria in neonates and infants from a tertiary care center, North India.

PURPOSE: Candida albicans is the major cause of fungal UTI in neonates and infants but nowadays non albicans Candida is also increasing and these are mostly multidrug resistant. So it's important to know the species of candidal UTI for the proper management. This study was undertaken to determine the Candida species distribution in UTI along with their susceptibility pattern and outcome in infants and neonates admitted in different wards and ICU of our hospital. We also assess the incidence rate of candiduria in ICUs. METHOD: Urine samples were collected from infants and neonates presented in pediatrics and neonatal ICU (intensive care units) and clinical wards with a clinical suspicion of candiduria and infants at risk of invasive candidiasis were also included in the study. Identification of Candida sp. was done by Gram's staining, germ tube test, chlamydospore formation on corn meal agar, color appearance on CHROM agar and also confirmed by MALDI-TOF Assay. Antifungal susceptibility was performed by using broth microdilution method as per the CLSI M27-A3/M27-S4. RESULT: Urine samples were received from 219 infants, and Candida was isolated from samples from 52 infants (isolation rate 23.75%), of which 30 were admitted in pediatric or neonatal ICU and 22 in the wards. The incidence rate of candiduria in ICU was 3.25%. Candida albicans was the most frequently isolated species from the samples of infants in the wards (13/22 i.e. 59%), while Candida tropicalis was most frequently isolated from samples of infants in the ICUs (13/30 i.e. 43.34%). Candida glabrata was the least commonly isolated species and was only encopuntered in the ICU. There was no discrepancy between the results of conventional methods of identification and MALDI-TOF. Antifungal susceptibility was performed for 18 randomly selected isolates. All were found to be susceptible to caspofungin, micafungin, itraconazole, voriconazole, fluconazole, amphotericin B. CONCLUSION: High suspicion of candiduria is needed especially in ICU admitted infants and identification of candida at species level along with the susceptibility pattern is important for the better management of patients.

Detection and Correction of Delocalization Errors for Electron and Hole Polarons Using Density-Corrected DFT.

Modeling polaron defects is an important aspect of computational materials science, but the description of unpaired spins in density functional theory (DFT) often suffers from delocalization error. To diagnose and correct the overdelocalization of spin defects, we report an implementation of density-corrected (DC-)DFT and its analytic energy gradient. In DC-DFT, an exchange-correlation functional is evaluated using a Hartree-Fock density, thus incorporating electron correlation while avoiding self-interaction error. Results for an electron polaron in models of titania and a hole polaron in Al-doped silica demonstrate that geometry optimization with semilocal functionals drives significant structural distortion, including the elongation of several bonds, such that subsequent single-point calculations with hybrid functionals fail to afford a localized defect even in cases where geometry optimization with the hybrid functional does localize the polaron. This has significant implications for traditional workflows in computational materials science, where semilocal functionals are often used for structure relaxation. DC-DFT calculations provide a mechanism to detect situations where delocalization error is likely to affect the results.

Emerging Aspergillus lentulus infections in India.

Here we report first two cases of invasive pulmonary aspergillosis caused by Aspergillus lentulus from India, in non-neutropenic, critically ill patients with chronic obstructive pulmonary disease (COPD).

Hidden Hemibonding in the Aqueous Hydroxyl Radical.

The existence of a two-center, three-electron hemibond in the first solvation shell of •OH(aq) has long been a matter of debate. The hemibond manifests in ab initio molecular dynamics simulations as a small-r feature in the oxygen radial distribution function (RDF) for H2O···â€¢OH, but that feature disappears when semilocal density functionals are replaced with hybrids, suggesting a self-interaction artifact. Using periodic simulations at the PBE0+D3 level, we demonstrate that the hemibond is actually still present (as evidenced by delocalization of the spin density) but is obscured by the hydrogen-bonded feature in the RDF due to a slight elongation of the hemibond. Computed electronic spectra for •OH(aq) are in excellent agreement with experiment and confirm that hemibond-like configurations play an outsized role in the spectroscopy due to an intense charge-transfer transition that is strongly attenuated in hydrogen-bonded configurations. Apparently, 25% exact exchange (as in PBE0) is insufficient to eliminate delocalization of unpaired spins.

Development of a Sensitive and Specific Novel qPCR Assay for Simultaneous Detection and Differentiation of Mucormycosis and Aspergillosis by Melting Curve Analysis.

Molecular diagnostic assays can expedite the diagnosis of fungal infections, and subsequently help in early interventions and appropriate management of patients. The aim of this study was to develop a single set of primers for a real-time quantitative polymerase chain reaction (qPCR) assay to detect and identify commonly reported, clinically relevant molds i.e., Aspergillus spp, Mucorales and Fusarium spp., up to genus level by melting curve analysis. This assay was evaluated in whole blood from patients with suspected invasive aspergillosis (IA), and in tissue biopsy, bronchoalveolar lavage (BAL) fluid and other site-specific samples from patients with suspected invasive mucormycosis (IM). The limit of detection (LoD) was determined as 10 copies/µl for all three molds. The mean coefficient of variation (CV) across all sets of intra- and inter-assay data was 0.63% (ranging from 0.42 to 1.56%), showing high reproducibility of the assay. Sensitivity and specificity of the assay were 93.3 and 97.1% respectively for diagnosis of IA, and 99.29 and 83.84% respectively for diagnosis of IM. Fusarium was not detected in any of the clinical samples included and the few laboratory confirmed cases of fusariosis did not meet the inclusion criteria of the study. Hence no ROC curve or cutoff value could be generated for the same. This newly developed qPCR assay therefore appears to be a promising tool in detection of IA and IM.

Role of hemibonding in the structure and ultraviolet spectroscopy of the aqueous hydroxyl radical.

The presence of a hemibond in the local solvation structure of the aqueous hydroxyl radical has long been debated, as its appearance in ab initio simulations based on density functional theory is sensitive to self-interaction error (favoring a two-center, three-electron hemibond) but also to finite-size effects. Simulations reported here use a mixed quantum mechanics/molecular mechanics (QM/MM) framework in a very large periodic simulation cell, in order to avoid finite-size artifacts and to facilitate testing of various density functionals, in order to probe the effects of delocalization error. The preponderance of hemibonded structures predicted by generalized gradient approximations persists in simulations using the hybrid functionals B3LYP and PBE0, but is reduced to a minor population if the fraction of exact exchange is increased to 50%. The hemibonded population is also small in simulations employing the long-range corrected functional LRC-ωPBE. Electronic spectra are computed using time-dependent density functional theory, and from these calculations emerges a consensus picture in which hemibonded configurations play an outsized role in the absorption spectrum, even when present as a minority species. An intense 1b2(H2O) → 2pπ(˙OH) charge-transfer transition in hemibonded configurations of the radical proves to be responsible for an absorption feature at 230 nm that is strongly shifted with respect to the gas-phase absorption at 307 nm, but this intense feature is substantially diminished in aqueous geometries where the hemibond is absent. Although not yet sufficient to quantitatively establish the population of hemibonded ˙OH(aq), these simulations do suggest that its presence is revealed by the strongly shifted ultraviolet absorption spectrum of the aqueous radical.

Ab Initio Investigation of the Resonance Raman Spectrum of the Hydrated Electron.

According to the conventional picture, the aqueous or "hydrated" electron, e-(aq), occupies an excluded volume (cavity) in the structure of liquid water. However, simulations with certain one-electron models predict a more delocalized spin density for the unpaired electron, with no distinct cavity structure. It has been suggested that only the latter (non-cavity) structure can explain the hydrated electron's resonance Raman spectrum, although this suggestion is based on calculations using empirical frequency maps developed for neat liquid water, not for e-(aq). All-electron ab initio calculations presented here demonstrate that both cavity and non-cavity models of e-(aq) afford significant red-shifts in the O-H stretching region. This effect is nonspecific and arises due to electron penetration into frontier orbitals of the water molecules. Only the conventional cavity model, however, reproduces the splitting of the H-O-D bend (in isotopically mixed water) that is observed experimentally and arises due to the asymmetric environments of the hydroxyl moieties in the electron's first solvation shell. We conclude that the cavity model of e-(aq) is more consistent with the measured resonance Raman spectrum than is the delocalized, non-cavity model, despite previous suggestions to the contrary. Furthermore, calculations with hybrid density functionals and with Hartree-Fock theory predict that non-cavity liquid geometries afford only unbound (continuum) states for an extra electron, whereas in reality this energy level should lie more than 3 eV below vacuum level. As such, the non-cavity model of e-(aq) appears to be inconsistent with available vibrational spectroscopy, photoelectron spectroscopy, and quantum chemistry.

Variational Formulation of the Generalized Many-Body Expansion with Self-Consistent Charge Embedding: Simple and Correct Analytic Energy Gradient for Fragment-Based ab Initio Molecular Dynamics.

The many-body expansion (MBE) and its extension to overlapping fragments, the generalized (G)MBE, constitute the theoretical basis for most fragment-based approaches for large-scale quantum chemistry. We reformulate the GMBE for use with embedding charges determined self-consistently from the fragment wave functions, in a manner that preserves the variational nature of the underlying self-consistent field method. As a result, the analytic gradient retains the simple "sum of fragment gradients" form that is often assumed in practice, sometimes incorrectly. This obviates (without approximation) the need to solve coupled-perturbed equations, and we demonstrate stable, fragment-based ab initio molecular dynamics simulations using this technique. Energy conservation fails when charge-response contributions to the Fock matrix are neglected, even while geometry optimizations and vibrational frequency calculations may yet be accurate. Stable simulations can be recovered by means of straightforward modifications introduced here, providing a general paradigm for fragment-based ab initio molecular dynamics.

Analytic gradient for the QM/MM-Ewald method using charges derived from the electrostatic potential: Theory, implementation, and application to ab initio molecular dynamics simulation of the aqueous electron.

We report an implementation of periodic boundary conditions for mixed quantum mechanics/molecular mechanics (QM/MM) simulations, in which atomic partial charges are used to represent periodic images of the QM region. These charges are incorporated into the Fock matrix in a manner that preserves the variational nature of the self-consistent field procedure, and their interactions with the MM charges are summed using the conventional Ewald technique. To ensure that the procedure is stable in arbitrary basis sets, the atomic charges are derived by least-squares fit to the electrostatic potential generated by the QM region. We formulate and implement analytic energy gradients for the QM/MM-Ewald method and demonstrate that stable molecular dynamics simulations are thereby obtained. As a proof-of-concept application, we perform QM/MM simulations of a hydrated electron in bulk liquid water at the level of Hartree-Fock theory plus empirical dispersion. These simulations demonstrate that the "cavity model" of the aqueous electron, in which the spin density of the anionic defect is localized within an excluded volume in the liquid, is stable at room temperature on a time scale of at least several picoseconds. These results validate cavity-forming pseudopotential models of e-(aq) that have previously been derived from static-exchange Hartree-Fock calculations, and cast doubt upon whether non-cavity-forming pseudopotentials are faithful to the underlying Hartree-Fock calculation from which they were obtained.