Recent Developments in Culture-Independent Fungal Diagnostics.

Culture-independent diagnostics for fungi potentially offer increased sensitivity and more rapid results relative to culture. Recent developments include new platforms for fungal cell wall antigen detection, commercially available targeted nucleic acid amplification tests, and the use of sequencing-based technologies for the detection and identification of a broad range of fungal pathogens. Although these tests are not without limitations, many more commercially available platforms now exist, and efforts to increase the standardization of laboratory-developed tests are ongoing.

Opportunities and challenges for the U.S. laboratory response to highly pathogenic avian influenza A(H5N1).

On March 25, 2024 an outbreak of highly pathogenic avian influenza (HPAI) A H5N1 was identified in dairy cows across multiple farms in the United States. Zoonotic cases originating in individuals with close contact to infected herds and poultry flocks have been subsequently identified. Spillover events such as this raise the specter of recent pandemics including COVID-19 and Mpox and may lead clinical laboratories to assess their capacity for diagnosis of HPAI H5N1.

A retrospective observational study of mNGS test utilization to examine the role of diagnostic stewardship at two academic medical centers.

Unlabelled: Given the cost and unclear clinical impact of metagenomic next-generation sequencing (mNGS), laboratory stewardship may improve utilization. This retrospective observational study examines mNGS results from two academic medical centers employing different stewardship approaches. Eighty mNGS orders [54 cerebrospinal fluid (CSF) and 26 plasma] were identified from 2019 to 2021 at the University of Washington (UW), which requires director-level approval for mNGS orders, and the University of Utah (Utah), which does not restrict ordering.

Repeat testing of mpox specimens with late CTs improves detection of potential false positive cases.

The mpox pandemic necessitated the rapid development of clinical assays for monkeypox virus detection. While the majority of mpox specimens have high viral loads with corresponding early cycle threshold (CT) values, reports have indicated some specimens with late CT values can represent false positive results. To mitigate this risk, the Centers for Disease Control and Prevention (CDC) published an advisory recommending repeat testing of all specimens with CT values ≥34.

Pulmonary Co-Infections Detected Premortem Underestimate Postmortem Findings in a COVID-19 Autopsy Case Series.

Bacterial and fungal co-infections are reported complications of coronavirus disease 2019 (COVID-19) in critically ill patients but may go unrecognized premortem due to diagnostic limitations. We compared the premortem with the postmortem detection of pulmonary co-infections in 55 fatal COVID-19 cases from March 2020 to March 2021. The concordance in the premortem versus the postmortem diagnoses and the pathogen identification were evaluated.

Clinical testing of pediatric mpox specimens: Unique features and challenges in a low prevalence population.

Background: Pediatric mpox cases comprise less than 0.3% of the total cases reported in the United States during the global 2022 outbreak. As a result, relatively little is known about the epidemiology or performance characteristics of clinical testing in this group.

Structures of Fission Yeast Inositol Pyrophosphate Kinase Asp1 in Ligand-Free, Substrate-Bound, and Product-Bound States.

Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP. IP dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg or Mn; IP, 5-IP, or 1,5-IP) and the other which was ligand-free.

Review of the Clinical Presentation, Pathology, Diagnosis, and Treatment of Leishmaniasis.

Leishmaniasis is a vector-borne infection caused by kinetoplastid protozoans in the genera Leishmania and Endotrypanum. The disease occurs worldwide in the tropics and subtropics and can be particularly burdensome in resource-limited settings. Diseases caused by leishmaniasis range in severity from mild cutaneous lesions to life-threatening visceral and disfiguring mucocutaneous illnesses.

Characterization of the Cytopathic Effects of Monkeypox Virus Isolated from Clinical Specimens and Differentiation from Common Viral Exanthems.

While the practice of viral culture has largely been replaced by nucleic acid amplification tests, circumstances still exist in which the availability of viral culture will allow for the diagnosis of infections not included in a provider's differential diagnosis. Here, we examine the cytopathic effects (CPE) and clinical data associated with 18 cases of monkeypox virus (MPXV) isolated from 19 clinical samples submitted for viral culture. During the study period, a total of 3,468 viral cultures were performed with herpes simplex virus (HSV) most commonly isolated (646/3,468; 18.

Clinical Performance and Trends during the First Two Months of Monkeypox Virus PCR Testing at Two United States Reference Labs.

Recently, a sustained human-to-human outbreak of monkeypox virus (MPXV), a member of the genus, which includes the etiologic agent of smallpox, has been documented in multiple nonendemic countries, including the United States. Prior to June 2022, testing in the United States was limited to public health labs and the Centers for Disease Control and Prevention. Following recognition of the scope of the outbreak, testing for MPXV has expanded into clinical laboratories.

Cokeromyces recurvatus Incidentally Found in a Patient with Gastric Outlet Obstruction.

Isolation of Cokeromyces recurvatus, a dimorphic mucormycete fungus, from clinical specimens poses a diagnostic challenge to physicians and laboratorians as this organism may represent a rare colonizer or true pathogen. Here, we report a case of Cokeromyces recurvatus present in a circumferential duodenal lesion. The patient is a 64-year-old with no past medical history, admitted with a three-week history of left upper quadrant abdominal pain.

Activities and Structure-Function Analysis of Fission Yeast Inositol Pyrophosphate (IPP) Kinase-Pyrophosphatase Asp1 and Its Impact on Regulation of Gene Expression.

Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP derepress the regulon while mutations that ablate IP synthesis are hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain.

Single nucleotide polymorphism rs 2070874 at Interleukin-4 is associated with increased risk of type 1 diabetes mellitus independently of human leukocyte antigens.

Introduction: Type 1 diabetes mellitus (T1DM) is characterized by autoimmune destruction of insulin-producing pancreatic beta (β-) cells. Previous studies suggested an imbalance between and pro- and anti-inflammatory cytokines exacerbates T1DM development.

Objectives: We aimed to test the hypothesis that patients with T1DM carry a higher frequency of regulatory genes associated with low levels of the anti-inflammatory cytokines interleukin-4 (IL-4), its receptor (IL-4R), and interleukin-10 (IL-10).

Cleavage-Polyadenylation Factor Cft1 and SPX Domain Proteins Are Agents of Inositol Pyrophosphate Toxicosis in Fission Yeast.

Inositol pyrophosphate (IPP) dynamics govern expression of the fission yeast phosphate homeostasis regulon via their effects on lncRNA-mediated transcription interference. The growth defects (ranging from sickness to lethality) elicited by fission yeast mutations that inactivate IPP pyrophosphatase enzymes are exerted via the agonistic effects of too much 1,5-IP8 on RNA 3'-processing and transcription termination. To illuminate determinants of IPP toxicosis, we conducted a genetic screen for spontaneous mutations that suppressed the sickness of Asp1 pyrophosphatase mutants.

IL-13 is a driver of COVID-19 severity.

Immune dysregulation is characteristic of the more severe stages of SARS-CoV-2 infection. Understanding the mechanisms by which the immune system contributes to COVID-19 severity may open new avenues to treatment. Here, we report that elevated IL-13 was associated with the need for mechanical ventilation in 2 independent patient cohorts.

Anti-SARS-CoV-2 Antibody Levels Measured by the AdviseDx SARS-CoV-2 Assay Are Concordant with Previously Available Serologic Assays but Are Not Fully Predictive of Sterilizing Immunity.

With the availability of widespread SARS-CoV-2 vaccination, high-throughput quantitative anti-spike protein serological testing will likely become increasingly important. Here, we investigated the performance characteristics of the recently FDA-authorized semiquantitative anti-spike protein AdviseDx SARS-CoV-2 IgG II assay compared to the FDA-authorized anti-nucleocapsid protein Abbott Architect SARS-CoV-2 IgG, Roche Elecsys anti-SARS-CoV-2-S, EuroImmun anti-SARS-CoV-2 enzyme-linked immunosorbent assay (ELISA), and GenScript surrogate virus neutralization assays and examined the humoral response associated with vaccination, natural protection, and vaccine breakthrough infection. The AdviseDx assay had a clinical sensitivity at 14 days after symptom onset or 10 days after PCR detection of 95.

Association of single-nucleotide polymorphisms in tumour necrosis factor and human leukocyte antigens genes with type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by progressive destruction of insulin-producing pancreatic beta cells. This multifactorial disease has a strong genetic component associated with the human leukocyte antigens (HLA) and non-HLA regions. In this study, we compared frequencies of HLA-DRB1 alleles and single-nucleotide polymorphisms (SNPs) associated the genes coding for: toll-like receptors (TLRs), tumour necrosis factor (TNF), interleukin-1 (IL-1), interleukin-1 receptor type 1 (IL-1R1), interleukin-1 receptor antagonist (IL-1RN), interleukin-2 (IL-2) and interleukin-12B (IL-12B), between T1D patients and healthy controls.

Generation of BK and JC Polyomavirus Defective Viral Genomes in Human Urine Samples Associated with Higher Viral Loads.

Defective viral genomes (DVGs) are parasitic viral sequences containing point mutations, deletions, or duplications that might interfere with replication. DVGs are often associated with viral passage at high multiplicities of infection in culture systems but have been increasingly reported in clinical specimens. To date however, only RNA viruses have been shown to contain DVGs in clinical specimens.

Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22.

Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3' processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core.

Distinct Cdk9-phosphatase switches act at the beginning and end of elongation by RNA polymerase II.

Reversible phosphorylation of Pol II and accessory factors helps order the transcription cycle. Here, we define two kinase-phosphatase switches that operate at different points in human transcription. Cdk9/cyclin T1 (P-TEFb) catalyzes inhibitory phosphorylation of PP1 and PP4 complexes that localize to 3' and 5' ends of genes, respectively, and have overlapping but distinct specificities for Cdk9-dependent phosphorylations of Spt5, a factor instrumental in promoter-proximal pausing and elongation-rate control.

Histopathology and ultrastructural findings of fatal COVID-19 infections in Washington State: a case series.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of an ongoing pandemic, with increasing deaths worldwide. To date, documentation of the histopathological features in fatal cases of the disease caused by SARS-CoV-2 (COVID-19) has been scarce due to sparse autopsy performance and incomplete organ sampling. We aimed to provide a clinicopathological report of severe COVID-19 cases by documenting histopathological changes and evidence of SARS-CoV-2 tissue tropism.

High-resolution profiling of human cytomegalovirus cell-free DNA in human plasma highlights its exceptionally fragmented nature.

Human cytomegalovirus (CMV) infections comprise a leading cause of newborn impairments worldwide and are pervasive concerns among the immunocompromised. Quantification of CMV viral loads is increasingly used to guide definitions of CMV disease but standardization of CMV quantitation remains problematic, mostly due to differences in qPCR amplicon sizes between clinical laboratories. Here, we used plasma cfDNA sequencing data from 2,208 samples sent for non-invasive prenatal aneuploidy screening to detect CMV and precisely measure the length of CMV fragments in human plasma.