Evaluation of 5 Polymerase Chain Reaction Assays for the Detection of Mpox Virus.

BACKGROUND: In 2022, the global dissemination of mpox virus (MPXV) outside endemic regions prompted the expansion of diagnostic testing worldwide. This study assesses the performance characteristics of 5 real-time polymerase chain reaction (PCR) assays in detecting MPXV during the 2022 outbreak. METHODS: Clinical specimens collected from patients across Ontario, Canada, were tested on the following assays: RealStar Orthopoxyvirus PCR and FlexStar Monkeypox virus PCR (Altona Diagnostics), Novaplex MPXV (Seegene), VIASURE Monkeypox virus Real Time PCR Reagents (CerTest Biotec), and a laboratory-developed test. Positive percent agreement (PPA), negative percent agreement (NPA), relative limit of detection (LOD), and precision were evaluated and MPXV lineages were determined using an amplicon-based whole-genome sequencing (WGS) assay. RESULTS: Swabs were collected from various anatomic sites (65 positive and 30 negative). All assays demonstrated 100% NPA (95% confidence interval, 88.4%/88.1%-100.0%), with PPA ranging from 92.2% (82.7%-97.4%) to 96.9% (89.3%-99.6%). LOD and precision were comparable across assays, with coefficient of variations <3%. WGS analysis identified 6 lineages, all belonging to subclade IIb. CONCLUSIONS: The assays exhibited excellent PPA, NPA, LOD, and precision. Ongoing performance monitoring is essential to detect assay escape mutants and ensure universal detection of evolving MPXV strains.

A Prolonged Outbreak of Human Adenovirus A31 (HAdV-A31) Infection on a Pediatric Hematopoietic Stem Cell Transplantation Ward with Whole Genome Sequencing Evidence of International Linkages.

A surge in hematopoietic stem cell transplantation (HSCT) human adenovirus A31 (HAdV-A31) infections was initially observed in late 2014/2015 at SickKids (SK) Hospital, Toronto, Canada. In response, enhanced laboratory monitoring for all adenovirus infections was conducted. Positive samples underwent genotyping, viral culture, and, in selected cases, whole-genome sequencing (WGS). HAdV-A31 specimens/DNA obtained from four international pediatric HSCT centers also underwent WGS. During the SK outbreak period (27 October 2014 to 31 October 2018), 17/20 HAdV-A31 isolates formed a distinct clade with 0 to 8 mutations between the closest neighbors. Surveillance before and after the outbreak detected six additional HAdV-A31 HSCT cases; three of the four sequenced cases clustered within the outbreak clade. Two SK outbreak isolates were identical to sequences from two patients in an outbreak in England. Three SK non-outbreak sequences also had high sequence similarity to strains from three international centers. Environmental PCR testing of the HSCT ward showed significant adenovirus contamination. Despite intense infection control efforts, we observed re-occurrence of infection with the outbreak strain. Severe but nonfatal infection was observed more commonly with HAdV-A31 compared to other genotypes, except HAdV-C1. Our findings strongly implicate nosocomial spread of HAdV-A31 over 10 years on a HSCT unit and demonstrate the value of WGS in defining and mapping the outbreak. Close linkages among strains in different countries suggest international dissemination, though the mechanism is undetermined. This large, extended outbreak emphasizes the pre-eminent role of HAdV-A31 in causing intractable pediatric HSCT outbreaks of severe illness worldwide.

COVID-19 Outbreak in an Urban Hemodialysis Unit.

RATIONALE & OBJECTIVE: Hemodialysis patients are at increased risk for coronavirus disease 2019 (COVID-19) transmission due in part to difficulty maintaining physical distancing. Our hemodialysis unit experienced a COVID-19 outbreak despite following symptom-based screening guidelines. We describe the course of the COVID-19 outbreak and the infection control measures taken for mitigation. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: 237 maintenance hemodialysis patients and 93 hemodialysis staff at a single hemodialysis center in Toronto, Canada. EXPOSURE: Universal screening of patients and staff for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). OUTCOMES: The primary outcome was detection of SARS-CoV-2 in nasopharyngeal samples from patients and staff using reverse transcriptase-polymerase chain reaction (RT-PCR). ANALYTICAL APPROACH: Descriptive statistics were used for clinical characteristics and the primary outcome. RESULTS: 11 of 237 (4.6%) hemodialysis patients and 11 of 93 (12%) staff members had a positive RT-PCR test result for SARS-CoV-2. Among individuals testing positive, 12 of 22 (55%) were asymptomatic at time of testing and 7 of 22 (32%) were asymptomatic for the duration of follow-up. One patient was hospitalized at the time of SARS-CoV-2 infection and 4 additional patients with positive test results were subsequently hospitalized. 2 (18%) patients required admission to the intensive care unit. After 30 days' follow-up, no patients had died or required mechanical ventilation. No hemodialysis staff required hospitalization. Universal droplet and contact precautions were implemented during the outbreak. Hemodialysis staff with SARS-CoV-2 infection were placed on home quarantine regardless of symptom status. Patients with SARS-CoV-2 infection, including asymptomatic individuals, were treated with droplet and contact precautions until confirmation of negative SARS-CoV-2 RT-PCR test results. Analysis of the outbreak identified 2 index cases with subsequent nosocomial transmission within the dialysis unit and in shared shuttle buses to the hemodialysis unit. LIMITATIONS: Single-center study. CONCLUSIONS: Universal SARS-CoV-2 testing and universal droplet and contact precautions in the setting of an outbreak appeared to be effective in preventing further transmission.

Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread.

Efferocytosis, the process by which dying or dead cells are removed by phagocytosis, has an important role in development, tissue homeostasis and innate immunity. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes, can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells by using the pore-forming toxin listeriolysin O (LLO) and two phospholipase C enzymes. Expression of the cell surface protein ActA allows L. monocytogenes to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. Here we show that protrusion formation is associated with plasma membrane damage due to LLO's pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 (encoded by the Timd4 gene) contributes to efficient cell-to-cell spread by L. monocytogenes in macrophages in vitro and growth of these bacteria is impaired in Timd4(-/-) mice. Thus, L. monocytogenes promotes its dissemination in a host by exploiting efferocytosis. Our results indicate that PS-targeted therapeutics may be useful in the fight against infections by L. monocytogenes and other bacteria that use similar strategies of cell-to-cell spread during infection.

The diaphanous-related formins promote protrusion formation and cell-to-cell spread of Listeria monocytogenes.

The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular pathogen whose virulence depends on its ability to spread from cell to cell within an infected host. Although the actin-related protein 2/3 (Arp2/3) complex is necessary and sufficient for Listeria actin tail assembly, previous studies suggest that other actin polymerization factors, such as formins, may participate in protrusion formation. Here, we show that Arp2/3 localized to only a minor portion of the protrusion. Moreover, treatment of L. monocytogenes-infected HeLa cells with a formin FH2-domain inhibitor significantly reduced protrusion length. In addition, the Diaphanous-related formins 1-3 (mDia1-3) localized to protrusions, and knockdown of mDia1, mDia2, and mDia3 substantially decreased cell-to-cell spread of L. monocytogenes. Rho GTPases are known to be involved in formin activation. Our studies also show that knockdown of several Rho family members significantly influenced bacterial cell-to-cell spread. Collectively, these findings identify a Rho GTPase-formin network that is critically involved in the cell-to-cell spread of L. monocytogenes.

What Is the Appropriate Meropenem MIC for Screening of Carbapenemase-Producing Enterobacteriaceae in Low-Prevalence Settings?

Infection with carbapenemase-producing Enterobacteriaceae (CPE) has been shown to cause significant illness among hospitalized patients. Given the paucity of treatment options, there is a critical need to stop the spread of CPE. However, screening for the presence of CPE in laboratory settings has been challenging. In order to assess the effectiveness of current CPE detection guidelines, we analyzed the meropenem MIC distribution for a large set of clinical Enterobacteriaceae isolates. A total of 1,022 isolates submitted to the Public Health Ontario Laboratories (PHOL) from January 2011 to March 2014 were examined. Only isolates displaying a meropenem or ertapenem MIC of ≥ 0.25 or ≥ 1 µg/ml, respectively, were included. Carbapenemase-positive isolates were identified by multiplex PCR. We identified 189 isolates positive for carbapenemases, which primarily comprised NDM, KPC, and OXA-48-like carbapenemases, and these isolates were largely Klebsiella spp., Escherichia coli, and Enterobacter spp. Interestingly, 14 to 20% of these isolates displayed meropenem MICs within the susceptible range on the basis of CLSI and EUCAST breakpoint interpretive criteria. While the majority of meropenem-susceptible CPE isolates were observed to be E. coli, meropenem susceptibility was not exclusive to any one species/genus or carbapenemase type. Application of CLSI screening recommendations captured only 86% of carbapenemase-producing isolates, whereas application of EUCAST recommendations detected 98.4% of CPE isolates. In a region with a low carbapenemase prevalence, meropenem-based screening approaches require a cutoff MIC near the epidemiological wild-type threshold in order to achieve nearly optimal CPE identification.

Variants in nicotinamide adenine dinucleotide phosphate oxidase complex components determine susceptibility to very early onset inflammatory bowel disease.

BACKGROUND & AIMS: The colitis observed in patients with very early onset inflammatory bowel disease (VEOIBD; defined as onset of disease at younger than 6 years of age) often resembles that of chronic granulomatous disease (CGD) in extent and features of colonic inflammation observed by endoscopy and histology. CGD is a severe immunodeficiency caused by defects in the genes that encode components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex. We investigated whether variants in genes that encode NADPH oxidase components affect susceptibility to VEOIBD using independent approaches. METHODS: We performed targeted exome sequencing of genes that encode components of NADPH oxidases (cytochrome b light chain and encodes p22(phox) protein; cytochrome b-245 or NADPH oxidase 2, and encodes Nox2 or gp91(phox); neutrophil cytosol factor 1 and encodes p47 (phox) protein; neutrophil cytosol factor 2 and encodes p67 (phox) protein; neutrophil cytosol factor 4 and encodes p40 (phox) protein; and Ras-related C3 botulinum toxin substrate 1 and 2) in 122 patients with VEOIBD diagnosed at The Hospital for Sick Children, University of Toronto, from 1994 through 2012. Gene variants were validated in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients with VEOIBD. In a second approach, we examined Tag single nucleotide polymorphisms in a subset of patients with VEOIBD in which the NOX2 NADPH oxidase genes sequence had been previously analyzed. We then looked for single nucleotide polymorphisms associated with the disease in an independent International Early Onset Pediatric IBD Cohort Study cohort of patients. We analyzed the functional effects of variants associated with VEOIBD. RESULTS: Targeted exome sequencing and Tag single nucleotide polymorphism genotyping identified 11 variants associated with VEOIBD; the majority of patients were heterozygous for these variants. Expression of these variants in cells either reduced oxidative burst or altered interactions among proteins in the NADPH oxidase complex. Variants in the noncoding regulatory and splicing elements resulted in reduced levels of proteins, or expression of altered forms of the proteins, in blood cells from VEOIBD patients. CONCLUSIONS: We found that VEOIBD patients carry heterozygous functional hypomorphic variants in components of the NOX2 NADPH oxidase complex. These do not cause overt immunodeficiency, but instead determine susceptibility to VEOIBD. Specific approaches might be developed to treat individual patients based on their genetic variant.

Mutations in tetratricopeptide repeat domain 7A result in a severe form of very early onset inflammatory bowel disease.

BACKGROUND & AIMS: Very early onset inflammatory bowel diseases (VEOIBD), including infant disorders, are a diverse group of diseases found in children younger than 6 years of age. They have been associated with several gene variants. Our aim was to identify the genes that cause VEOIBD. METHODS: We performed whole exome sequencing of DNA from 1 infant with severe enterocolitis and her parents. Candidate gene mutations were validated in 40 pediatric patients and functional studies were carried out using intestinal samples and human intestinal cell lines. RESULTS: We identified compound heterozygote mutations in the Tetratricopeptide repeat domain 7 (TTC7A) gene in an infant from non-consanguineous parents with severe exfoliative apoptotic enterocolitis; we also detected TTC7A mutations in 2 unrelated families, each with 2 affected siblings. TTC7A interacts with EFR3 homolog B to regulate phosphatidylinositol 4-kinase at the plasma membrane. Functional studies demonstrated that TTC7A is expressed in human enterocytes. The mutations we identified in TTC7A result in either mislocalization or reduced expression of TTC7A. Phosphatidylinositol 4-kinase was found to co-immunoprecipitate with TTC7A; the identified TTC7A mutations reduced this binding. Knockdown of TTC7A in human intestinal-like cell lines reduced their adhesion, increased apoptosis, and decreased production of phosphatidylinositol 4-phosphate. CONCLUSIONS: In a genetic analysis, we identified loss of function mutations in TTC7A in 5 infants with VEOIBD. Functional studies demonstrated that the mutations cause defects in enterocytes and T cells that lead to severe apoptotic enterocolitis. Defects in the phosphatidylinositol 4-kinase-TTC7A-EFR3 homolog B pathway are involved in the pathogenesis of VEOIBD.

Evaluation of the utility and cost of secondary confirmatory testing for Neisseria gonorrhoeae identification from culture.

Current guideline recommends the use of two identification methods for Neisseria gonorrhoeae. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) is now used for primary identification and may be sufficient for definitive identification of N. gonorrhoeae. The performance of three secondary tests (BactiCard, RapID NH and NET test) were compared using 45 bacterial isolates, including 37 Neisseria species. These secondary tests demonstrated diminished specificity (67% - 88%) for N. gonorrhoeae compared with MALDI-TOF. Additionally, data from six clinical microbiology laboratories was used to compare confirmatory test costs and the agreement of results with MALDI-TOF. Discrepancies were documented for 9.4% of isolates, though all isolates (n= 288) identified by MALDI-TOF as N. gonorrhoeae were confirmed by the reference laboratory. These data demonstrate that MALDI-TOF alone is sufficient for N. gonorrhoeae identification, as secondary did not add diagnostic value but do add costs to the testing process.

A mouse model of airway disease: oncostatin M-induced pulmonary eosinophilia, goblet cell hyperplasia, and airway hyperresponsiveness are STAT6 dependent, and interstitial pulmonary fibrosis is STAT6 independent.

Oncostatin M (OSM), a pleiotropic cytokine of the gp130 cytokine family, has been implicated in chronic allergic inflammatory and fibrotic disease states associated with tissue eosinophilia. Mouse (m)OSM induces airway eosinophilic inflammation and interstitial pulmonary fibrosis in vivo and regulates STAT6 activation in vitro. To determine the requirement of STAT6 in OSM-induced effects in vivo, we examined wild-type (WT) and STAT6-knockout (STAT6(-/-)) C57BL/6 mouse lung responses to transient ectopic overexpression of mOSM using an adenoviral vector (AdmOSM). Intratracheal AdmOSM elicited persistent eosinophilic lung inflammation that was abolished in STAT6(-/-) mice. AdmOSM also induced pronounced pulmonary remodeling characterized by goblet cell hyperplasia and parenchymal interstitial fibrosis. Goblet cell hyperplasia was STAT6 dependent; however, parenchymal interstitial fibrosis was not. OSM also induced airway hyperresponsiveness in WT mice that was abolished in STAT6(-/-) mice. OSM stimulated an inflammatory signature in the lungs of WT mice that demonstrated STAT6-dependent regulation of Th2 cytokines (IL-4, IL-13), chemokines (eotaxin-1/2, MCP-1, keratinocyte chemoattractant), and extracellular matrix modulators (tissue inhibitor of matrix metalloproteinase-1, matrix metalloproteinase-13), but STAT6-independent regulation of IL-4Rα, total lung collagen, collagen-1A1, -1A2 mRNA, and parenchymal collagen and α smooth muscle actin accumulation. Thus, overexpression of mOSM induces STAT6-dependent pulmonary eosinophilia, mucous/goblet cell hyperplasia, and airway hyperresponsiveness but STAT6-independent mechanisms of lung tissue extracellular matrix accumulation. These results also suggest that eosinophil or neutrophil accumulation in mouse lungs is not required for OSM-induced lung parenchymal collagen deposition and that OSM may have unique roles in the pathogenesis of allergic and fibrotic lung disease.

NADPH oxidase complex and IBD candidate gene studies: identification of a rare variant in NCF2 that results in reduced binding to RAC2.

OBJECTIVE: The NOX2 NADPH oxidase complex produces reactive oxygen species and plays a critical role in the killing of microbes by phagocytes. Genetic mutations in genes encoding components of the complex result in both X-linked and autosomal recessive forms of chronic granulomatous disease (CGD). Patients with CGD often develop intestinal inflammation that is histologically similar to Crohn's colitis, suggesting a common aetiology for both diseases. The aim of this study is to determine if polymorphisms in NOX2 NADPH oxidase complex genes that do not cause CGD are associated with the development of inflammatory bowel disease (IBD). METHODS: Direct sequencing and candidate gene approaches were used to identify susceptibility loci in NADPH oxidase complex genes. Functional studies were carried out on identified variants. Novel findings were replicated in independent cohorts. RESULTS: Sequence analysis identified a novel missense variant in the neutrophil cytosolic factor 2 (NCF2) gene that is associated with very early onset IBD (VEO-IBD) and subsequently found in 4% of patients with VEO-IBD compared with 0.2% of controls (p=1.3×10(-5), OR 23.8 (95% CI 3.9 to 142.5); Fisher exact test). This variant reduced binding of the NCF2 gene product p67(phox) to RAC2. This study found a novel genetic association of RAC2 with Crohn's disease (CD) and replicated the previously reported association of NCF4 with ileal CD. CONCLUSION: These studies suggest that the rare novel p67(phox) variant results in partial inhibition of oxidase function and are associated with CD in a subgroup of patients with VEO-IBD; and suggest that components of the NADPH oxidase complex are associated with CD.

Post-traumatic Fungal Keratitis and Endophthalmitis Caused by Coniochaeta Hoffmannii with Late Recurrence following Therapeutic Full-thickness Penetrating Keratoplasty.

BACKGROUND: To report a rare case of fungal keratitis and endophthalmitis due to Coniochaeta hoffmannii. METHODS: Case report. RESULTS: A 71-year-old immunocompetent male sustained a corneal laceration, traumatic cataract, and retinal detachment due to penetrating injury from a nail pulled from a wooden deck. The patient's postoperative course was complicated by infectious keratitis. Fungal cultures, DNA sequencing and analysis of the internal transcribed spacer sequence confirmed Coniochaeta hoffmannii. Topical and oral voriconazole treatments were initiated; however, due to impending perforation, a therapeutic corneal transplant was required. One year later, the patient developed a new corneal infiltrate at the graft-host junction: Corneal scrapings were culture positive for Coniochaeta hoffmannii. This was treated with topical and intrastromal voriconazole along with oral itraconazole 200 mg once daily for 8 months. CONCLUSIONS: Coniochaeta hoffmannii may cause recalcitrant keratitis and endophthalmitis, which required longstanding antifungal treatment.

Prevalence of Low-Frequency, Antiviral Resistance Variants in SARS-CoV-2 Isolates in Ontario, Canada, 2020-2023.

Importance: Nirmatrelvir-ritonavir is an oral antiviral medication that improves outcomes in SARS-CoV-2 infections. However, there is concern that antiviral resistance will develop and that these viruses could be selected for after treatment. Objective: To determine the prevalence of low-frequency SARS-CoV-2 variants in patient samples that could be selected for by nirmatrelvir-ritonavir. Design, Setting, and Participants: This retrospective cohort study was conducted at 4 laboratories that serve community hospitals, academic tertiary care centers, and COVID-19 assessment centers in Ontario, Canada. Participants included symptomatic or asymptomatic patients who tested positive for SARS-CoV-2 virus and submitted virus samples for diagnostic testing between March 2020 and January 2023. Exposure: SARS-CoV-2 infection. Main Outcomes and Measures: Samples with sufficient viral load underwent next-generation genome sequencing to identify low-frequency antiviral resistance variants that could not be identified through conventional sequencing. Results: This study included 78 866 clinical samples with next-generation whole-genome sequencing data for SARS-CoV-2. Low-frequency variants in the viral nsp5 gene were identified in 128 isolates (0.16%), and no single variant associated with antiviral resistance was predominate. Conclusions and Relevance: This cohort study of low-frequency variants resistant to nirmatrelvir-ritonavir found that these variants were very rare in samples from patients with SARS-CoV-2, suggesting that selection of these variants by nirmatrelvir-ritonavir following the initiation of treatment may also be rare. Surveillance efforts that involve sequencing of viral isolates should continue to monitor for novel resistance variants as nirmatrelvir-ritonavir is used more broadly.

A Novel Approach to Detect IDH Point Mutations in Gliomas Using Nanopore Sequencing: Test Validation for the Clinical Laboratory.

The use of standard next-generation sequencing technologies to detect key mutations in IDH genes for glioma diagnosis imposes several challenges, including high capital cost and turnaround delays associated with the need for batch testing. For both glioma testing and testing in other tumor types where highly specific mutation identification is required, the high-throughput nature of next-generation sequencing limits the feasibility of using it as a primary approach in clinical laboratories. We hypothesized that third-generation nanopore sequencing by Oxford Nanopore Technologies has the capability to overcome these limitations. This study aimed to develop and validate a nanopore-based IDH mutation detection assay for clinical practice using glioma formalin-fixed, paraffin-embedded (FFPE) tissue. Glioma FFPE (n = 66) samples with confirmed IDH gene mutational status were sequenced on the MinION device using an amplicon-based approach. All cases were concordant when compared with the reference results. Limit of blank and limit of detection for the variant allele fraction were 1.5% and 3.3%, respectively, at 500× read depth per gene. Total sequencing cost per sample was CAD$50 to CAD$134 with results being available in 9 to 15 hours. These findings demonstrate that nanopore-sequencing technology can be leveraged to develop low-cost, high-performance clinical sequencing-based assays with quick turnaround times to support the detection of targeted mutations in FFPE tumor tissue.

Coronavirus disease 2019 (COVID-19) outbreak on an in-patient medical unit associated with unrecognized exposures in common areas-Epidemiological and whole-genome sequencing investigation.

OBJECTIVE: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hospital outbreaks have been common and devastating during the coronavirus disease 2019 (COVID-19) pandemic. Understanding SARS-CoV-2 transmission in these environments is critical for preventing and managing outbreaks. DESIGN: Outbreak investigation through epidemiological mapping and whole-genome sequencing phylogeny. SETTING: Hospital in-patient medical unit outbreak in Toronto, Canada, from November 2020 to January 2021. PARTICIPANTS: The outbreak involved 8 patients and 10 staff and was associated with 3 patient deaths. RESULTS: Patients being cared for in geriatric chairs at the nursing station were at high risk for both acquiring and transmitting SARS-CoV-2 to other patients and staff. Furthermore, given the informal nature of these transmissions, they were not initially recognized, which led to further transmission and missing the opportunity for preventative COVID-19 therapies. CONCLUSIONS: During outbreak prevention and management, the risk of informal patient care settings, such as geriatric chairs, should be considered. During high-risk periods or during outbreaks, efforts should be made to care for patients in their rooms when possible.

Single nucleotide polymorphisms that increase expression of the guanosine triphosphatase RAC1 are associated with ulcerative colitis.

BACKGROUND & AIMS: RAC1 is a guanosine triphosphatase that has an evolutionarily conserved role in coordinating immune defenses, from plants to mammals. Chronic inflammatory bowel diseases are associated with dysregulation of immune defenses. We studied the role of RAC1 in inflammatory bowel diseases using human genetic and functional studies and animal models of colitis. METHODS: We used a candidate gene approach to HapMap-Tag single nucleotide polymorphisms in a discovery cohort; findings were confirmed in 2 additional cohorts. RAC1 messenger RNA expression was examined from peripheral blood cells of patients. Colitis was induced in mice with conditional disruption of Rac1 in phagocytes by administration of dextran sulfate sodium. RESULTS: We observed a genetic association between RAC1 with ulcerative colitis in a discovery cohort, 2 independent replication cohorts, and in combined analysis for the single nucleotide polymorphisms rs10951982 (P(combined UC) = 3.3 × 10(-8), odds ratio = 1.43 [95% confidence interval: 1.26-1.63]) and rs4720672 (P(combined UC) = 4.7 × 10(-6), odds ratio = 1.36 [95% confidence interval: 1.19-1.58]). Patients with inflammatory bowel disease who had the rs10951982 risk allele had increased expression of RAC1 compared to those without this allele. Conditional disruption of Rac1 in macrophage and neutrophils of mice protected against dextran sulfate sodium-induced colitis. CONCLUSIONS: Human studies and knockout mice demonstrated a role for the guanosine triphosphatase RAC1 in the development of ulcerative colitis; increased expression of RAC1 was associated with susceptibility to colitis.

Eosinophils are dispensable for allergic remodeling and immunity in a model of house dust mite-induced airway disease.

RATIONALE: Current thinking accredits eosinophils with preeminent contributions to allergic airway responses, including a major role in the development of airway remodeling, a process thought to significantly contribute to airway dysfunction. However, direct evidence in support of this notion is limited and often controversial. OBJECTIVES: We elucidated the requirement for eosinophils in the generation of allergic sensitization, airway inflammation, and remodeling in a model involving chronic respiratory exposure to house dust mite (HDM). METHODS: We used three methods to selectively eliminate eosinophils, a depleting antibody (anti-CCR3), and two strains of eosinophil-deficient mice (ΔdblGATA and the transgenic line PHIL). MEASUREMENTS AND MAIN RESULTS: Anti-CCR3 treatment markedly reduced pulmonary eosinophilia (> 80%) over the course of HDM exposure but had no effect on the remaining inflammatory response, the extent of lung Th2 cells, or the development of remodeling-associated changes, including subepithelial collagen deposition and smooth muscle thickening. In addition, we observed that, despite the absence of eosinophils, HDM-exposed GATA mice mounted robust airway and lung inflammation and hyperresponsiveness and showed a remodeling response equivalent to that observed in wild-type mice. Moreover, these mice had similar serum HDM-specific IgE levels and Th2-associated splenocyte cytokine production as HDM-exposed wild-type control mice. Similar observations were made in PHIL eosinophil-deficient mice subjected to chronic HDM exposure, although slight decreases in airway mononuclear cells, but not lung Th2 cells, and remodeling were noted. CONCLUSIONS: Collectively, these data demonstrate that, at variance with the prevailing paradigm, eosinophils play negligible roles in the generation of HDM-induced allergic immunity and airway remodeling.

Comparison of SARS-CoV-2 Viral Loads in the Nasal Mucosa of Patients Infected With BA.1, BA.2, or BA.5 Omicron Lineages.

Lower viral loads were observed in the upper respiratory tract of patients infected with BA.1, whereas patients infected with BA.2 and BA.5 had comparable viral loads to those seen with Alpha or Delta. This suggests that viral loads are likely not responsible for the increased transmission of the Omicron lineages.

Atypical Clinical Presentation of Monkeypox Complicated by Myopericarditis.

We present a case of monkeypox infection in a man presenting with genital and labial ulcers, followed by submandibular lymphadenopathy, fever, and constitutional symptoms. His course was complicated by myopericarditis and an ongoing pleomorphic skin eruption. Viral deoxyribonucleic acid was detected by polymerase chain reaction in skin swabs, nasopharyngeal swab, saliva, and semen.