Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2024 Update by the Infectious Diseases Society of America (IDSA) and the American Society for Microbiology (ASM).

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician and the microbiologists who provide enormous value to the health care team. This document, developed by experts in both adult and pediatric laboratory and clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. Sections are divided into anatomic systems, including Bloodstream Infections and Infections of the Cardiovascular System, Central Nervous System Infections, Ocular Infections, Soft Tissue Infections of the Head and Neck, Upper Respiratory Infections, Lower Respiratory Tract infections, Infections of the Gastrointestinal Tract, Intraabdominal Infections, Bone and Joint Infections, Urinary Tract Infections, Genital Infections, and Skin and Soft Tissue Infections; or into etiologic agent groups, including arboviral Infections, Viral Syndromes, and Blood and Tissue Parasite Infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also addressed. There is redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a reference to guide physicians in choosing tests that will aid them to diagnose infectious diseases in their patients.

First Case Report of an Unusual Fungus (Sporopachydermia lactativora) Associated with a Pulmonary Infection in a Drug Injection User.

In contrast to a robust literature on known pathogenic fungi such as Cryptococcus and Aspergillus species that cause pulmonary infections, reports of the uncommon genus Sporopachydermia causing infections are very limited. We present the first case report describing the fungus, Sporopachydermia lactativora as a likely cause of pneumonia in a patient with a history of polysubstance abuse and injection drug use (IDU). The patient recovered following antifungal treatment. The organism was recovered from a blood culture, 3 days post collection. Although CHROMagar was of little value, only yeast-like organisms were observed on cornmeal agar. The organism was not in the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry database. Definitive identification was achieved using the ribosomal DNA (rDNA) sequence analysis by targeting the ITS1 (internal transcribed spacer 1) region. This case report is intended to promote awareness of this fungus as a potential pathogen, by providing new information that has not yet been reported in the literature, and prompts physician awareness to suspect a fungal infection when managing patients with a history of IDU as a potential source of unique environmental organisms not previously encountered, warranting more comprehensive diagnosis and treatment options.

The reliability of saliva for the detection of SARS-CoV-2 in symptomatic and asymptomatic patients: Insights on the diagnostic performance and utility for COVID-19 screening.

Current literature has focused on testing saliva in symptomatic patients, and little information is available regarding saliva performance in asymptomatic severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. We compared paired saliva and nasopharyngeal swabs (NPS) collected from 33 symptomatic and 12 asymptomatic known SARS-CoV-2-positive patients. Saliva had an overall sensitivity of 59%, a specificity of 95%, and a negative predictive value of 98%. Saliva demonstrated higher sensitivity in symptomatic (80%) vs. asymptomatic individuals (36%) (P = 0.006), and in high-risk (symptomatic, febrile and/or with comorbidities) (82%) vs. low-risk (asymptomatic, afebrile, and no comorbidities) (22%) patients (P = 0.0002). Cycle threshold (Ct) values in NPS specimens were higher in saliva-negative vs. saliva-positive cases (P = 0.02 and <0.001). Overall, these findings show that despite saliva's low sensitivity in asymptomatic SARS-CoV-2 infections, it can detect infections with lower Ct values and a potentially higher chance of viral transmission. Additional studies are warranted to fully evaluate saliva as a screening test for coronavirus disease-2019.

Clinical Laboratory Biosafety Gaps: Lessons Learned from Past Outbreaks Reveal a Path to a Safer Future.

Patient care and public health require timely, reliable laboratory testing. However, clinical laboratory professionals rarely know whether patient specimens contain infectious agents, making ensuring biosafety while performing testing procedures challenging. The importance of biosafety in clinical laboratories was highlighted during the 2014 Ebola outbreak, where concerns about biosafety resulted in delayed diagnoses and contributed to patient deaths. This review is a collaboration between subject matter experts from large and small laboratories and the federal government to evaluate the capability of clinical laboratories to manage biosafety risks and safely test patient specimens. We discuss the complexity of clinical laboratories, including anatomic pathology, and describe how applying current biosafety guidance may be difficult as these guidelines, largely based on practices in research laboratories, do not always correspond to the unique clinical laboratory environments and their specialized equipment and processes. We retrospectively describe the biosafety gaps and opportunities for improvement in the areas of risk assessment and management; automated and manual laboratory disciplines; specimen collection, processing, and storage; test utilization; equipment and instrumentation safety; disinfection practices; personal protective equipment; waste management; laboratory personnel training and competency assessment; accreditation processes; and ethical guidance. Also addressed are the unique biosafety challenges successfully handled by a Texas community hospital clinical laboratory that performed testing for patients with Ebola without a formal biocontainment unit. The gaps in knowledge and practices identified in previous and ongoing outbreaks demonstrate the need for collaborative, comprehensive solutions to improve clinical laboratory biosafety and to better combat future emerging infectious disease outbreaks.

Digital imaging for reading of direct rapid antibiotic susceptibility tests from positive blood cultures.

Delaying effective antibiotic therapy is a major cause of sepsis-associated mortality. The EUCAST rapid antibiotic susceptibility test (RAST) is performed from positive blood cultures to provide rapid results. Disc diffusion tests inoculated with positive blood culture broth are read at 4, 6, and 8 h and interpreted against species and time-specific criteria. Potential problems are the possibility of missing specific reading times for tests and slower growth in incubators that are frequently opened. The current study aimed to assess if digital visualization by the BD Kiestra™ total laboratory automation system is suitable for reading RASTs by capturing images at the correct times and retaining them for review. Utilizing the Kiestra™ InoqulA, 100 µl of positive blood culture broth was lawn-inoculated onto Mueller-Hinton agar and incubated at 35 °C for automated digital zone measurement at 4, 6, and 8 h. Aliquots from 135 positive blood cultures were tested against EUCAST-recommended and other drugs and assessed for readability of digital images. Microdilution MICs were determined in parallel to RASTs. All isolates except 7/10 enterococci yielded images of suitable quality for zone measurement. Of the 641 digitally read tests for other organisms, 207 (32.3%) were readable in 4 h, 555 (86.6%) in 6 h, and 641 (100%) in 8 h. For tests included in EUCAST criteria, 92.1% provided categorical agreement with microdilution MICs. Digital image reading of RASTs is a potentially viable, inexpensive tool for providing rapid susceptibility results which can help reduce sepsis-associated mortality.

Automated preparation for identification and antimicrobial susceptibility testing: evaluation of a research use only prototype, the BD Kiestra IdentifA/SusceptA system.

OBJECTIVE: The current BD Kiestra™ total laboratory automation (TLA) system automates specimen inoculation, incubation, and digital visualization of cultures prior to initiation of manual or semi-automated identification (ID) and antimicrobial susceptibility testing (AST). The current study aimed to compare the performance, in a clinical setting, of a fully automated research-use-only prototype, BD Kiestra™ IdentifA/SusceptA (automated system), to our current BD Kiestra™ TLA which utilizes manual or semi-automated IDs and ASTs (current system). METHODS: Clinical samples yielding significant growth after processing by the BD Kiestra™ TLA were tested in parallel for ID and AST by both systems. IDs and ASTs were determined by Bruker matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry and BD Phoenix, respectively, with data stored and managed in the BD EpiCenter™. The automated system used a common inoculum preparation for both tests, whereas the current system used separate inocula. Results were compared to assess agreement between the systems. RESULTS: On initial testing, 89% of IDs (466/523) and 92.4% of IDs (484/523) for the automated and current ID systems, respectively, yielded acceptable MALDI-TOF log scores of ≥1.7. On repeat testing, the respective acceptable scores were 97.1% (508/523) and 98.1% (513/523). For initial ASTs, the automated and current systems yielded 97.5% categorical agreement for 7325 drug-organism tests. After omitting discrepant MICs that differed by only one dilution and categorical discrepancies that were not reproducible, 0.2% unresolved discrepancies remained thus (99.8% categorical agreement). CONCLUSIONS: The automated prototype is suitable for development into technology that will provide clinical microbiology laboratories with significant advantages such as improved efficiency, standardization, reproducibility, reduced technical error and greater safety.

TeraChem: Accelerating electronic structure and ab initio molecular dynamics with graphical processing units.

Developed over the past decade, TeraChem is an electronic structure and ab initio molecular dynamics software package designed from the ground up to leverage graphics processing units (GPUs) to perform large-scale ground and excited state quantum chemistry calculations in the gas and the condensed phase. TeraChem's speed stems from the reformulation of conventional electronic structure theories in terms of a set of individually optimized high-performance electronic structure operations (e.g., Coulomb and exchange matrix builds, one- and two-particle density matrix builds) and rank-reduction techniques (e.g., tensor hypercontraction). Recent efforts have encapsulated these core operations and provided language-agnostic interfaces. This greatly increases the accessibility and flexibility of TeraChem as a platform to develop new electronic structure methods on GPUs and provides clear optimization targets for emerging parallel computing architectures.

Reducing Blood Culture Contamination Rates: Experiences of Four Hospital Systems.

INTRODUCTION: Blood cultures (BCs) frequently become contaminated during the pre-analytic phase of collection leading to downstream ramifications. We present a summary of performance improvement (PI) interventions provided by four hospital systems and common factors that contributed to decreased blood culture contamination (BCC) rates. METHODS: Each hospital independently formed a multidisciplinary team and action plan for implementation of their intervention, focusing on the use of educational and training tools. Their goal was to significantly decrease their BCC rates. Pre- and post-intervention data were compared during the sustainment period to determine their success. RESULTS: All hospitals met their goals of post-intervention BCC rates and with most achieving and sustaining BCC rates ≤ 1.0-2.0%. CONCLUSION: Our report highlights how four hospitals independently achieved their objective to decrease their BCC rate with the support of a multidisciplinary team. We propose a benchmark for BCC rates of 1.5 to < 2.0% as achievable and sustainable.

A Laboratory Medicine Best Practices Systematic Review and Meta-analysis of Nucleic Acid Amplification Tests (NAATs) and Algorithms Including NAATs for the Diagnosis of Clostridioides (Clostridium) difficile in Adults.

The evidence base for the optimal laboratory diagnosis of Clostridioides (Clostridium) difficile in adults is currently unresolved due to the uncertain performance characteristics and various combinations of tests. This systematic review evaluates the diagnostic accuracy of laboratory testing algorithms that include nucleic acid amplification tests (NAATs) to detect the presence of C. difficile The systematic review and meta-analysis included eligible studies (those that had PICO [population, intervention, comparison, outcome] elements) that assessed the diagnostic accuracy of NAAT alone or following glutamate dehydrogenase (GDH) enzyme immunoassays (EIAs) or GDH EIAs plus C. difficile toxin EIAs (toxin). The diagnostic yield of NAAT for repeat testing after an initial negative result was also assessed. Two hundred thirty-eight studies met inclusion criteria. Seventy-two of these studies had sufficient data for meta-analysis. The strength of evidence ranged from high to insufficient. The uses of NAAT only, GDH-positive EIA followed by NAAT, and GDH-positive/toxin-negative EIA followed by NAAT are all recommended as American Society for Microbiology (ASM) best practices for the detection of the C. difficile toxin gene or organism. Meta-analysis of published evidence supports the use of testing algorithms that use NAAT alone or in combination with GDH or GDH plus toxin EIA to detect the presence of C. difficile in adults. There is insufficient evidence to recommend against repeat testing of the sample using NAAT after an initial negative result due to a lack of evidence of harm (i.e., financial, length of stay, or delay of treatment) as specified by the Laboratory Medicine Best Practices (LMBP) systematic review method in making such an assessment. Findings from this systematic review provide clarity to diagnostic testing strategies and highlight gaps, such as low numbers of GDH/toxin/PCR studies, in existing evidence on diagnostic performance, which can be used to guide future clinical research studies.

Activity of Cefepime-Zidebactam against Multidrug-Resistant (MDR) Gram-Negative Pathogens.

This study compared the activity of cefepime + zidebactam (FEP-ZID) and selected currently available antibacterial agents against a panel of multidrug-resistant (MDR) clinical isolates chosen to provide an extreme challenge for antibacterial activity. FEP⁻ZID had a very broad and potent in vitro spectrum of activity, and was highly active against many MDR isolates of Enterobacterales, Pseudomonas aeruginosa, and Acinetobacter baumannii. Notably, it inhibited isolates producing carbapenemases of Ambler classes A, B, and D, and P. aeruginosa isolates with multiple resistance mechanisms including combinations of upregulated efflux, diminished or non-functional OprD porins, and AmpC overproduction. Its clinical role will be determined initially by the breakpoints assigned to it, comparison studies with other investigational ß-lactamase inhibitor combinations, and ultimately by the developing body of therapeutic outcome data.

A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology.

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

A Guide to Utilization of the Microbiology Laboratory for Diagnosis of Infectious Diseases: 2018 Update by the Infectious Diseases Society of America and the American Society for Microbiology.

The critical nature of the microbiology laboratory in infectious disease diagnosis calls for a close, positive working relationship between the physician/advanced practice provider and the microbiologists who provide enormous value to the healthcare team. This document, developed by experts in laboratory and adult and pediatric clinical medicine, provides information on which tests are valuable and in which contexts, and on tests that add little or no value for diagnostic decisions. This document presents a system-based approach rather than specimen-based approach, and includes bloodstream and cardiovascular system infections, central nervous system infections, ocular infections, soft tissue infections of the head and neck, upper and lower respiratory infections, infections of the gastrointestinal tract, intra-abdominal infections, bone and joint infections, urinary tract infections, genital infections, and other skin and soft tissue infections; or into etiologic agent groups, including arthropod-borne infections, viral syndromes, and blood and tissue parasite infections. Each section contains introductory concepts, a summary of key points, and detailed tables that list suspected agents; the most reliable tests to order; the samples (and volumes) to collect in order of preference; specimen transport devices, procedures, times, and temperatures; and detailed notes on specific issues regarding the test methods, such as when tests are likely to require a specialized laboratory or have prolonged turnaround times. In addition, the pediatric needs of specimen management are also emphasized. There is intentional redundancy among the tables and sections, as many agents and assay choices overlap. The document is intended to serve as a guidance for physicians in choosing tests that will aid them to quickly and accurately diagnose infectious diseases in their patients.

Ultrafast isomerization in acetylene dication after carbon K-shell ionization.

Ultrafast proton migration and isomerization are key processes for acetylene and its ions. However, the mechanism for ultrafast isomerization of acetylene [HCCH]2+ to vinylidene [H2CC]2+ dication remains nebulous. Theoretical studies show a large potential barrier ( > 2 eV) for isomerization on low-lying dicationic states, implying picosecond or longer isomerization timescales. However, a recent experiment at a femtosecond X-ray free-electron laser suggests sub-100 fs isomerization. Here we address this contradiction with a complete theoretical study of the dynamics of acetylene dication produced by Auger decay after X-ray photoionization of the carbon atom K shell. We find no sub-100 fs isomerization, while reproducing the salient features of the time-resolved Coulomb imaging experiment. This work resolves the seeming contradiction between experiment and theory and also calls for careful interpretation of structural information from the widely applied Coulomb momentum imaging method.The timescale of isomerization in molecules involving ultrafast migration of constituent atoms is difficult to measure. Here the authors report that sub-100 fs isomerization time on acetylene dication in lower electronic states is not possible and point to misinterpretation of recent experimental results.

α-CASSCF: An Efficient, Empirical Correction for SA-CASSCF To Closely Approximate MS-CASPT2 Potential Energy Surfaces.

Because of its computational efficiency, the state-averaged complete active-space self-consistent field (SA-CASSCF) method is commonly employed in nonadiabatic ab initio molecular dynamics. However, SA-CASSCF does not effectively recover dynamical correlation. As a result, there can be qualitative differences between SA-CASSCF potential energy surfaces (PESs) and more accurate reference surfaces computed using multistate complete active space second-order perturbation theory (MS-CASPT2). Here we introduce an empirical correction to SA-CASSCF that scales the splitting between individual states and the state-averaged energy. We call this the α-CASSCF method, and we show here that it significantly improves the accuracy of relative energies and PESs compared with MS-CASPT2 for the chromophores of green fluorescent and photoactive yellow proteins. As such, this method may prove to be quite valuable for nonadiabatic dynamics.

A direct-compatible formulation of the coupled perturbed complete active space self-consistent field equations on graphical processing units.

We recently developed an algorithm to compute response properties for the state-averaged complete active space self-consistent field method (SA-CASSCF) that capitalized on sparsity in the atomic orbital basis. Our original algorithm was limited to treating small to moderate sized active spaces, but the recent development of graphical processing unit (GPU) based direct-configuration interaction algorithms provides an opportunity to extend this to large active spaces. We present here a direct-compatible version of the coupled perturbed equations, enabling us to compute response properties for systems treated with arbitrary active spaces (subject to available memory and computation time). This work demonstrates that the computationally demanding portions of the SA-CASSCF method can be formulated in terms of seven fundamental operations, including Coulomb and exchange matrix builds and their derivatives, as well as, generalized one- and two-particle density matrix and σ vector constructions. As in our previous work, this algorithm exhibits low computational scaling and is accelerated by the use of GPUs, making possible optimizations and nonadiabatic dynamics on systems with O(1000) basis functions and O(100) atoms, respectively.

GPU-Accelerated State-Averaged Complete Active Space Self-Consistent Field Interfaced with Ab Initio Multiple Spawning Unravels the Photodynamics of Provitamin D3.

Excited-state molecular dynamics is essential to the study of photochemical reactions, which occur under nonequilibrium conditions. However, the computational cost of such simulations has often dictated compromises between accuracy and efficiency. The need for an accurate description of both the molecular electronic structure and nuclear dynamics has historically stymied the simulation of medium- to large-size molecular systems. Here, we show how to alleviate this problem by combining ab initio multiple spawning (AIMS) for the nuclear dynamics and GPU-accelerated state-averaged complete active space self-consistent field (SA-CASSCF) for the electronic structure. We demonstrate the new approach by first-principles SA-CASSCF/AIMS nonadiabatic dynamics simulation of photoinduced electrocyclic ring-opening in the 51-atom provitamin D3 molecule.

Coproduction of KPC-18 and VIM-1 Carbapenemases by Enterobacter cloacae: Implications for Newer ß-Lactam-ß-Lactamase Inhibitor Combinations.

Enterobacter cloacae strain G6809 with reduced susceptibility to carbapenems was identified from a patient in a long-term acute care hospital in Kentucky. G6809 belonged to sequence type (ST) 88 and carried two carbapenemase genes, bla(KPC-18) and bla(VIM-1). Whole-genome sequencing localized bla(KPC-18) to the chromosome and bla(VIM-1) to a 58-kb plasmid. The strain was highly resistant to ceftazidime-avibactam. Insidious coproduction of metallo-ß-lactamase with KPC-type carbapenemase has implications for the use of next-generation ß-lactam-ß-lactamase inhibitor combinations.

An atomic orbital-based formulation of analytical gradients and nonadiabatic coupling vector elements for the state-averaged complete active space self-consistent field method on graphical processing units.

We recently presented an algorithm for state-averaged complete active space self-consistent field (SA-CASSCF) orbital optimization that capitalizes on sparsity in the atomic orbital basis set to reduce the scaling of computational effort with respect to molecular size. Here, we extend those algorithms to calculate the analytic gradient and nonadiabatic coupling vectors for SA-CASSCF. Combining the low computational scaling with acceleration from graphical processing units allows us to perform SA-CASSCF geometry optimizations for molecules with more than 1000 atoms. The new approach will make minimal energy conical intersection searches and nonadiabatic dynamics routine for molecular systems with O(10(2)) atoms.