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.

Tau and Aß42 in lavage fluid of pneumonia patients are associated with end-organ dysfunction: A prospective exploratory study.

BACKGROUND: Bacterial pneumonia and sepsis are both common causes of end-organ dysfunction, especially in immunocompromised and critically ill patients. Pre-clinical data demonstrate that bacterial pneumonia and sepsis elicit the production of cytotoxic tau and amyloids from pulmonary endothelial cells, which cause lung and brain injury in naïve animal subjects, independent of the primary infection. The contribution of infection-elicited cytotoxic tau and amyloids to end-organ dysfunction has not been examined in the clinical setting. We hypothesized that cytotoxic tau and amyloids are present in the bronchoalveolar lavage fluid of critically ill patients with bacterial pneumonia and that these tau/amyloids are associated with end-organ dysfunction. METHODS: Bacterial culture-positive and culture-negative mechanically ventilated patients were recruited into a prospective, exploratory observational study. Levels of tau and Aß42 in, and cytotoxicity of, the bronchoalveolar lavage fluid were measured. Cytotoxic tau and amyloid concentrations were examined in comparison with patient clinical characteristics, including measures of end-organ dysfunction. RESULTS: Tau and Aß42 were increased in culture-positive patients (n = 49) compared to culture-negative patients (n = 50), independent of the causative bacterial organism. The mean age of patients was 52.1 ± 16.72 years old in the culture-positive group and 52.78 ± 18.18 years old in the culture-negative group. Males comprised 65.3% of the culture-positive group and 56% of the culture-negative group. Caucasian culture-positive patients had increased tau, boiled tau, and Aß42 compared to both Caucasian and minority culture-negative patients. The increase in cytotoxins was most evident in males of all ages, and their presence was associated with end-organ dysfunction. CONCLUSIONS: Bacterial infection promotes the generation of cytotoxic tau and Aß42 within the lung, and these cytotoxins contribute to end-organ dysfunction among critically ill patients. This work illuminates an unappreciated mechanism of injury in critical illness.

Differential activation of programmed cell death in patients with severe SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe lower airway disease and death in a subset of patients. Knowledge on the relative contribution of programmed cell death (PCD) to lung pathology is limited to few human autopsy studies with small sample size/scope, in vitro cell culture, and experimental model systems. In this study, we sought to identify, localize, and quantify activation of apoptosis, ferroptosis, pyroptosis, and necroptosis in FFPE lung tissues from patients that died from severe SARS-CoV-2 infection (n = 28) relative to uninfected controls (n = 13). Immunofluorescence (IF) staining, whole-slide imaging, and Image J software was used to localize and quantify expression of SARS-CoV-2 nucleoprotein and the following PCD protein markers: cleaved Caspase-3, pMLKL, cleaved Gasdermin D, and CD71, respectively. IF showed differential activation of each PCD pathway in infected lungs and dichotomous staining for SARS-CoV-2 nucleoprotein enabling distinction between high (n = 9) vs low viral burden (n = 19). No differences were observed in apoptosis and ferroptosis in SARS-CoV-2 infected lungs relative to uninfected controls. However, both pyroptosis and necroptosis were significantly increased in SARS-CoV-2-infected lungs. Increased pyroptosis was observed in SARS-CoV-2 infected lungs, irrespective of viral burden, suggesting an inflammation-driven mechanism. In contrast, necroptosis exhibited a very strong positive correlation with viral burden (R2 = 0.9925), suggesting a direct SARS-CoV-2 mediated effect. These data indicate a possible novel mechanism for viral-mediated necroptosis and a potential role for both lytic programmed cell death pathways, necroptosis and pyroptosis, in mediating infection outcome.

Priorities and Progress in Gram-positive Bacterial Infection Research by the Antibacterial Resistance Leadership Group: A Narrative Review.

The Antibacterial Resistance Leadership Group (ARLG) has prioritized infections caused by gram-positive bacteria as one of its core areas of emphasis. The ARLG Gram-positive Committee has focused on studies responding to 3 main identified research priorities: (1) investigation of strategies or therapies for infections predominantly caused by gram-positive bacteria, (2) evaluation of the efficacy of novel agents for infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci, and (3) optimization of dosing and duration of antimicrobial agents for gram-positive infections. Herein, we summarize ARLG accomplishments in gram-positive bacterial infection research, including studies aiming to (1) inform optimal vancomycin dosing, (2) determine the role of dalbavancin in MRSA bloodstream infection, (3) characterize enterococcal bloodstream infections, (4) demonstrate the benefits of short-course therapy for pediatric community-acquired pneumonia, (5) develop quality of life measures for use in clinical trials, and (6) advance understanding of the microbiome. Future studies will incorporate innovative methodologies with a focus on interventional clinical trials that have the potential to change clinical practice for difficult-to-treat infections, such as MRSA bloodstream infections.

A conceptual framework for nomenclatural stability and validity of medically important fungi: a proposed global consensus guideline for fungal name changes supported by ABP, ASM, CLSI, ECMM, ESCMID-EFISG, EUCAST-AFST, FDLC, IDSA, ISHAM, MMSA, and MSGERC.

The rapid pace of name changes of medically important fungi is creating challenges for clinical laboratories and clinicians involved in patient care. We describe two sources of name change which have different drivers, at the species versus the genus level. Some suggestions are made here to reduce the number of name changes. We urge taxonomists to provide diagnostic markers of taxonomic novelties. Given the instability of phylogenetic trees due to variable taxon sampling, we advocate to maintain genera at the largest possible size. Reporting of identified species in complexes or series should where possible comprise both the name of the overarching species and that of the molecular sibling, often cryptic species. Because the use of different names for the same species will be unavoidable for many years to come, an open access online database of the names of all medically important fungi, with proper nomenclatural designation and synonymy, is essential. We further recommend that while taxonomic discovery continues, the adaptation of new name changes by clinical laboratories and clinicians be reviewed routinely by a standing committee for validation and stability over time, with reference to an open access database, wherein reasons for changes are listed in a transparent way.

Coronary Stent Abscess in the Setting of Arteriovenous Graft Infection following COVID-19: An Autopsy Case Report.

While rare, coronary stent infections present with significant mortality-with most infections and further complications occurring within months of percutaneous coronary intervention (PCI). Here, we discuss a post-COVID-19 patient who presented approximately one year after PCI for declotting of an arteriovenous graft (AVG). Upon admission, the patient was found to be bacteremic with multilobar pneumonia and an infection of the AVG. Empiric antibiotics were started, and blood cultures were subsequently positive for MRSA. Removal of the AVG was unsuccessful, and two days after admission, the patient passed. Autopsy revealed a perivascular abscess in the RCA near the origin of the stent with a ground section of the RCA with stent revealing abundant calcific atherosclerosis and marked necrosis of the artery wall. The cause of death was determined to be sepsis complicating coronary artery disease and chronic renal failure.

Interferon-dependent signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections, including COVID-19-related acute respiratory distress syndrome, although its contribution to disease pathogenesis remains poorly understood. Blood and airway immune cells from 52 patients with severe COVID-19 were phenotyped by flow cytometry. Samples and clinical data were collected at 2 separate time points to assess changes during ICU stay. Blockade of type I interferon and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) signaling was performed in vitro to determine their contribution to viral clearance in A2 neutrophils. We identified 2 neutrophil subpopulations (A1 and A2) in the airway compartment, where loss of the A2 subset correlated with increased viral burden and reduced 30-day survival. A2 neutrophils exhibited a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation, with consequent reduced viral catabolism, providing the first discrete mechanism to our knowledge of type I interferon signaling in neutrophils. The identification of this neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Immunity to pathogenic fungi in the eye.

Fusarium, Aspergillus and Candida are important fungal pathogens that cause visual impairment and blindness in the USA and worldwide. This review will summarize the epidemiology and clinical features of corneal infections and discuss the immune and inflammatory responses that play an important role in clinical disease. In addition, we describe fungal virulence factors that are required for survival in infected corneas, and the activities of neutrophils in fungal killing, tissue damage and cytokine production.

Type I interferon-dependent IFIT3 signaling is critical for viral clearance in airway neutrophils.

Neutrophilic inflammation characterizes several respiratory viral infections including COVID-19-related ARDS, although its contribution to disease pathogenesis remains poorly understood. Here, we identified two neutrophil subpopulations (A1 and A2) in the airway compartment of 52 severe COVID-19 subjects, where loss of the A2 subset correlated with increased viral burden and reduced 30-days survival. A2 neutrophils showcased a discrete antiviral response with an increased interferon signature. Blockade of type I interferon attenuated viral clearance in A2 neutrophils and downregulated IFIT3 and key catabolic genes, demonstrating direct antiviral neutrophil function. Knockdown of IFIT3 in A2 neutrophils led to loss of IRF3 phosphorylation with consequent reduced viral catabolism, providing the first discrete mechanism of type I interferon signaling in neutrophils. The identification of this novel neutrophil phenotype and its association with severe COVID-19 outcomes emphasizes its likely importance in other respiratory viral infections and potential for new therapeutic approaches in viral illness.

Mucociliary transport deficiency and disease progression in Syrian hamsters with SARS-CoV-2 infection.

Substantial clinical evidence supports the notion that ciliary function in the airways is important in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, the extent or nature of impairment of mucociliary transport (MCT) in in vivo models remains unknown. We hypothesize that SARS-CoV-2 infection results in MCT deficiency in the airways of golden Syrian hamsters that precedes pathological injury in lung parenchyma. Micro-optical coherence tomography was used to quantitate functional changes in the MCT apparatus. Both genomic and subgenomic viral RNA pathological and physiological changes were monitored in parallel. We show that SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 days postinfection (dpi) in hamsters, principally due to 79% diminished airway coverage of motile cilia. Correlating quantitation of physiological, virological, and pathological changes reveals steadily descending infection from the upper airways to lower airways to lung parenchyma within 7 dpi. Our results indicate that functional deficits of the MCT apparatus are a key aspect of COVID-19 pathogenesis, may extend viral retention, and could pose a risk factor for secondary infection. Clinically, monitoring abnormal ciliated cell function may indicate disease progression. Therapies directed toward the MCT apparatus deserve further investigation.

A single intranasal administration of AdCOVID protects against SARS-CoV-2 infection in the upper and lower respiratory tracts.

The coronavirus disease 2019 (COVID-19) pandemic has illustrated the critical need for effective prophylactic vaccination to prevent the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Intranasal vaccination is an attractive approach for preventing COVID-19 as the nasal mucosa is the site of initial SARS-CoV-2 entry and viral replication prior to aspiration into the lungs. We previously demonstrated that a single intranasal administration of a candidate adenovirus type 5-vectored vaccine encoding the receptor-binding domain of the SARS-CoV-2 spike protein (AdCOVID) induced robust immunity in both the airway mucosa and periphery, and completely protected K18-hACE2 mice from lethal SARS-CoV-2 challenge. Here we show that a single intranasal administration of AdCOVID limits viral replication in the nasal cavity of K18-hACE2 mice. AdCOVID also induces sterilizing immunity in the lungs of mice as reflected by the absence of infectious virus. Finally, AdCOVID prevents SARS-CoV-2 induced pathological damage in the lungs of mice. These data show that AdCOVID not only limits viral replication in the respiratory tract, but it also prevents virus-induced inflammation and immunopathology following SARS-CoV-2 infection.

COVID-19 Vaccination and Remdesivir are Associated With Protection From New or Increased Levels of Donor-Specific Antibodies Among Kidney Transplant Recipients Hospitalized With COVID-19.

Alloimmune responses in kidney transplant (KT) patients previously hospitalized with COVID-19 are understudied. We analyzed a cohort of 112 kidney transplant recipients who were hospitalized following a positive SARS-CoV-2 test result during the first 20 months of the COVID-19 pandemic. We found a cumulative incidence of 17% for the development of new donor-specific antibodies (DSA) or increased levels of pre-existing DSA in hospitalized SARS-CoV-2-infected KT patients. This risk extended 8 months post-infection. These changes in DSA status were associated with late allograft dysfunction. Risk factors for new or increased DSA responses in this KT patient cohort included the presence of circulating DSA pre-COVID-19 diagnosis and time post-transplantation. COVID-19 vaccination prior to infection and remdesivir administration during infection were each associated with decreased likelihood of developing a new or increased DSA response. These data show that new or enhanced DSA responses frequently occur among KT patients requiring admission with COVID-19 and suggest that surveillance, vaccination, and antiviral therapies may be important tools to prevent alloimmunity in these individuals.

COVID-19 Causes Ciliary Dysfunction as Demonstrated by Human Intranasal Micro-Optical Coherence Tomography Imaging.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2), causative agent of coronavirus disease 2019 (COVID-19), binds via ACE2 receptors, highly expressed in ciliated cells of the nasal epithelium. Micro-optical coherence tomography (µOCT) is a minimally invasive intranasal imaging technique that can determine cellular and functional dynamics of respiratory epithelia at 1-µm resolution, enabling real time visualization and quantification of epithelial anatomy, ciliary motion, and mucus transport. We hypothesized that respiratory epithelial cell dysfunction in COVID-19 will manifest as reduced ciliated cell function and mucociliary abnormalities, features readily visualized by µOCT. Symptomatic outpatients with SARS-CoV-2 aged ≥ 18 years were recruited within 14 days of symptom onset. Data was interpreted for subjects with COVID-19 (n=13) in comparison to healthy controls (n=8). Significant reduction in functional cilia, diminished ciliary beat frequency, and abnormal ciliary activity were evident. Other abnormalities included denuded epithelium, presence of mucus rafts, and increased inflammatory cells. Our results indicate that subjects with mild but symptomatic COVID-19 exhibit functional abnormalities of the respiratory mucosa underscoring the importance of mucociliary health in viral illness and disease transmission. Ciliary imaging enables investigation of early pathogenic mechanisms of COVID-19 and may be useful for evaluating disease progression and therapeutic response.

Maternal and Perinatal Outcomes Associated With the Omicron Variant of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infection.

Two years into the coronavirus disease 2019 (COVID-19) pandemic, we have now seen three main variant waves. We performed a retrospective cohort study of all pregnant patients with COVID-19 at our institution from March 22, 2020, to February 26, 2022, to evaluate disease severity and perinatal outcomes among the variants. Patients were categorized as pre-Delta (March 22, 2020-May 31, 2021), Delta (July 1, 2021-December 15, 2021), or Omicron (December 16, 2021- February 26, 2022) based on variant tracking from the Centers for Disease Control and Prevention and genotype sequencing at our institution. There were fewer cases of severe-critical disease (1.8% Omicron vs 13.3% pre-Delta and 24.1% Delta) and adverse perinatal outcomes during the Omicron wave compared with the pre-Delta and Delta waves.

Meta-analysis of the robustness of COVID-19 diagnostic kit performance during the early pandemic.

BACKGROUND: Accurate detection of SARS-CoV-2 is necessary to mitigate the COVID-19 pandemic. However, the test reagents and assay platforms are varied and may not be sufficiently robust to diagnose COVID-19. METHODS: We reviewed 85 studies (21 530 patients), published from five regions of the world, to highlight issues involved in the diagnosis of COVID-19 in the early phase of the pandemic. All relevant articles, published up to 31 May 2020, in PubMed, BioRiXv, MedRiXv and Google Scholar, were included. We evaluated the qualitative (9749 patients) and quantitative (10 355 patients) performance of RT-PCR and serologic diagnostic tests for real-world samples, and assessed the concordance (5538 patients) between test performance in meta-analyses. Synthesis of results was done using random effects modelling and bias was evaluated according to QUADAS-2 guidelines. RESULTS: The RT-PCR tests exhibited heterogeneity in the primers and reagents used. Of 1957 positive RT-PCR COVID-19 participants, 1585 had positive serum antibody (IgM±IgG) tests (sensitivity 0.81, 95% CI 0.66 to 0.90). While 3509 of 3581 participants RT-PCR negative for COVID-19 were found negative by serology testing (specificity 0.98, 95% CI 0.94 to 0.99). The chemiluminescent immunoassay exhibited the highest sensitivity, followed by ELISA and lateral flow immunoassays. Serology tests had higher sensitivity and specificity for laboratory approval than for real-world reporting data. DISCUSSION: The robustness of the assays/platforms is influenced by variability in sampling and reagents. Serological testing complements and may minimise false negative RT-PCR results. Lack of standardised assay protocols in the early phase of pandemic might have contributed to the spread of COVID-19.

Mucociliary Transport Deficiency and Disease Progression in Syrian Hamsters with SARS-CoV-2 Infection.

Substantial clinical evidence supports the notion that ciliary function in the airways plays an important role in COVID-19 pathogenesis. Although ciliary damage has been observed in both in vitro and in vivo models, consequent impaired mucociliary transport (MCT) remains unknown for the intact MCT apparatus from an in vivo model of disease. Using golden Syrian hamsters, a common animal model that recapitulates human COVID-19, we quantitatively followed the time course of physiological, virological, and pathological changes upon SARS-CoV-2 infection, as well as the deficiency of the MCT apparatus using micro-optical coherence tomography, a novel method to visualize and simultaneously quantitate multiple aspects of the functional microanatomy of intact airways. Corresponding to progressive weight loss up to 7 days post-infection (dpi), viral detection and histopathological analysis in both the trachea and lung revealed steadily descending infection from the upper airways, as the main target of viral invasion, to lower airways and parenchymal lung, which are likely injured through indirect mechanisms. SARS-CoV-2 infection caused a 67% decrease in MCT rate as early as 2 dpi, largely due to diminished motile ciliation coverage, but not airway surface liquid depth, periciliary liquid depth, or cilia beat frequency of residual motile cilia. Further analysis indicated that the fewer motile cilia combined with abnormal ciliary motion of residual cilia contributed to the delayed MCT. The time course of physiological, virological, and pathological progression suggest that functional deficits of the MCT apparatus predispose to COVID-19 pathogenesis by extending viral retention and may be a risk factor for secondary infection. As a consequence, therapies directed towards the MCT apparatus deserve further investigation as a treatment modality.

Diagnosis of Clostridioides difficile infection by analysis of volatile organic compounds in breath, plasma, and stool: A cross-sectional proof-of-concept study.

Clostridioides difficile infection (CDI) is an important infectious cause of antibiotic-associated diarrhea, with significant morbidity and mortality. Current diagnostic algorithms are based on identifying toxin by enzyme immunoassay (EIA) and toxin gene by real-time polymerase chain reaction (PCR) in patients with diarrhea. EIA's sensitivity is poor, and PCR, although highly sensitive and specific, cannot differentiate infection from colonization. An ideal test that incorporates microbial factors, host factors, and host-microbe interaction might characterize true infection, and assess prognosis and recurrence. The study of volatile organic compounds (VOCs) has the potential to be an ideal diagnostic test. The presence of VOCs accounts for the characteristic odor of stool in CDI but their presence in breath and plasma has not been studied yet. A cross-sectional proof-of-concept study analyzing VOCs using selected ion flow tube mass spectrometry (SIFT-MS) was done on breath, stool, and plasma of patients with clinical features and positive PCR for CDI (cases) and compared with patients with clinical features but a negative PCR (control). Our results showed that VOC patterns in breath, stool, and plasma, had good accuracy [area under the receiver operating characteristic curve (ROC) 93%, 86%, and 91%, respectively] for identifying patients with CDI.