Prospective Genomic Surveillance Reveals Cryptic MRSA Outbreaks with Local to International Origins among NICU Patients.

Methicillin-resistant Staphylococcus aureus (MRSA) infections cause substantive morbidity and mortality in neonates. Using publicly available resources from the National Center of Biotechnology Information (NCBI) and Food and Drug Administration's (FDA) GalaxyTrakr pipeline, we illustrate the dynamics of MRSA colonization and infection in neonates. Over 217 days of prospective surveillance, analyses revealed concurrent MRSA transmission chains affecting 11 of 17 MRSA-colonized patients (65%), with two clusters that demonstrated intervals of more than a month among the appearance of isolates. All MRSA infected neonates (n = 3) showed previous colonization with the infecting strain. GalaxyTrakr clustering of the NICU strains, in the context of 21,521 international isolates deposited in NCBI's Pathogen Detection Resource, revealed NICU isolates to be distinct from adult MRSA strains seen locally and internationally. Clustering of the NICU strains within an international context enhanced the resolution of strain clusters and supported the rule-out of suspected, local transmission events within the NICU. Analyses also identified sequence type 1535 isolates, emergent in the Middle East, carrying a unique SCCmec with fusC and aac(6')-Ie/aph(2'')-1a that provided a multidrug-resistant phenotype. NICU genomic pathogen surveillance, leveraging public repositories and outbreak detection tools, supports rapid identification of cryptic MRSA clusters, and can inform infection prevention interventions for this vulnerable patient population. Results demonstrate that sporadic infections in the NICU may be indicative of hidden chains of asymptomatic transmission best identified with sequenced-based approaches.

Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step.

The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples (n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.

Rigidoporus corticola Colonization and Invasive Fungal Disease in Immunocompromised Patients, United States.

We report 2 cases of Rigidoporus corticola (Oxyporus corticola) infection in humans in the United States. Clinical manifestations consisted of angioinvasive fungal sinusitis in 1 patient and pulmonary intracavitary fungus ball in the other patient. These cases illustrate previously undescribed clinicopathologic manifestations of infection by this filamentous basidiomycete in humans.

Daily, oral FMT for long-term maintenance therapy in ulcerative colitis: results of a single-center, prospective, randomized pilot study.

BACKGROUND: Fecal microbiota transplantation (FMT) is a promising new strategy in the treatment of Inflammatory Bowel Disease, but long-term delivery systems are lacking. This randomized study was designed as a safety and feasibility study of long-term FMT in subjects with mild to moderate UC using frozen, encapsulated oral FMT (cFMT). METHODS: Subjects were randomized 1:1 to receive FMT induction by colonoscopy, followed by 12 weeks of daily oral administration of frozen encapsulated cFMT or sham therpay. Subjects were followed for 36 weeks and longitudenal clinical assessments included multiple subjective and objective markers of disease severity. Ribosomal 16S bacterial sequencing was used to assess donor-induced changes in the gut microbiota. Changes in T regulatory (Treg) and mucosal associated invariant T (MAIT) cell populations were evaluated by flow cytometry as an exploratory endpoint. RESULTS: Twelve subjects with active UC were randomized: 6 subjects completed the full 12-week course of FMT plus cFMT, and 6 subjects received sham treatment by colonic installation and longitudinal oral placebo capules. Chronic administration of cFMT was found to be safe and well-tolerated but home storage concerns exist. Protocol adherence was high, and none of the study subjects experienced FMT-associated treatment emergent adverse events. Two subjects that received cFMT achieved clinical remission versus none in the placebo group (95% CI = 0.38-infinity, p = 0.45). cFMT was associated with sustained donor-induced shifts in fecal microbial composition. Changes in MAIT cell cytokine production were observed in cFMT recipients and correlated with treatment response. CONCLUSION: These pilot data suggest that daily encapsulated cFMT may extend the durability of index FMT-induced changes in gut bacterial community structure and that an association between MAIT cell cytokine production and clinical response to FMT may exist in UC populations. Oral frozen encapsulated cFMT is a promising FMT delivery system and may be preferred for longterm treatment strategies in UC and other chronic diseases but further evaluations will have to address home storage concerns. Larger trials should be done to explore the benefits of cFMT and to determine its long-term impacts on the colonic microbiome. TRIAL REGISTRATION: ClinicalTrials.gov (NCT02390726). Registered 17 March 2015, https://clinicaltrials.gov/ct2/show/NCT02390726?term=NCT02390726&draw=2&rank=1 .

Identification of novel loci controlling inflammatory bowel disease susceptibility utilizing the genetic diversity of wild-derived mice.

Inflammatory bowel disease (IBD) is a complex disorder that imposes a growing health burden. Multiple genetic associations have been identified in IBD, but the mechanisms underlying many of these associations are poorly understood. Animal models are needed to bridge this gap, but conventional laboratory mouse strains lack the genetic diversity of human populations. To more accurately model human genetic diversity, we utilized a panel of chromosome (Chr) substitution strains, carrying chromosomes from the wild-derived and genetically divergent PWD/PhJ (PWD) strain on the commonly used C57BL/6J (B6) background, as well as their parental B6 and PWD strains. Two models of IBD were used, TNBS- and DSS-induced colitis. Compared with B6 mice, PWD mice were highly susceptible to TNBS-induced colitis, but resistant to DSS-induced colitis. Using consomic mice, we identified several PWD-derived loci that exhibited profound effects on IBD susceptibility. The most pronounced of these were loci on Chr1 and Chr2, which yielded high susceptibility in both IBD models, each acting at distinct phases of the disease. Leveraging transcriptomic data from B6 and PWD immune cells, together with a machine learning approach incorporating human IBD genetic associations, we identified lead candidate genes, including Itga4, Pip4k2a, Lcn10, Lgmn, and Gpr65.

The utility of human papillomavirus testing in young women with atypical glandular cells on pap test.

OBJECTIVE: This study aimed to gain information regarding the follow-up diagnoses and human papillomavirus (HPV) status of women younger than 35 years diagnosed with atypical glandular cells (AGCs) on Pap test. MATERIALS AND METHODS: This is a retrospective observational study in which the cytopathology files at Fletcher Allen Health Care were reviewed from 2000 to 2013 for the diagnoses of AGC in women younger than 35 years. Subsequent pathology reports and HPV testing results were obtained. Significant lesions were defined as cervical intraepithelial neoplasia (CIN) 2 or 3, invasive squamous cell carcinoma, adenocarcinoma in situ, or adenocarcinoma. RESULTS: One hundred six women younger than 35 years with an AGC Pap diagnosis and subsequent follow-up were identified. Significant lesions were diagnosed in 44.3% of the women (47); the majority (55.3%, 26 patients) of which were classified as CIN 2 or 3. Adenocarcinoma in situ was diagnosed in 27.7% of the cases (13). A diagnosis of both CIN 2 or 3 and adenocarcinoma in situ was made in 14.9% of the cases (7). One patient (2.1%) was diagnosed with endometrial adenocarcinoma. The HPV status was identified in 36.8% of the women (39): 69.2% (27) was HPV positive, and 30.8% (12) was HPV negative. Fifty-five percent of HPV-positive women were diagnosed with a significant lesion upon follow-up. No known HPV-negative women were diagnosed with a significant lesion. CONCLUSIONS: Human papillomavirus testing may be useful in risk stratifying young women with AGC on Pap test because they are at risk of having an HPV-positive cervical lesion.

Variant mutation in SARS-CoV-2 nucleocapsid enhances viral infection via altered genomic encapsidation.

The evolution of SARS-CoV-2 variants and their respective phenotypes represents an important set of tools to understand basic coronavirus biology as well as the public health implications of individual mutations in variants of concern. While mutations outside of Spike are not well studied, the entire viral genome is undergoing evolutionary selection, particularly the central disordered linker region of the nucleocapsid (N) protein. Here, we identify a mutation (G215C), characteristic of the Delta variant, that introduces a novel cysteine into this linker domain, which results in the formation of a disulfide bond and a stable N-N dimer. Using reverse genetics, we determined that this cysteine residue is necessary and sufficient for stable dimer formation in a WA1 SARS-CoV-2 background, where it results in significantly increased viral growth both in vitro and in vivo. Finally, we demonstrate that the N:G215C virus packages more nucleocapsid per virion and that individual virions are larger, with elongated morphologies.

Recent advances in enterotoxin vaccine adjuvants.

Enterotoxin adjuvants have been researched for their ability to promote immunity to co-delivered antigens. Outside of cholera vaccines, however, these proteins have yet to be included in any currently licensed vaccines. They include molecules derived from the bacterial toxins of Vibrio cholerae, cholera toxin, or Escherichia coli, heat-labile toxin, such as detoxified mutants or subunits. This class of adjuvants is distinguished by their delivery possibilities, which include parenteral injection, skin applications, or direct mucosal administration by oral, sublingual, or nasal routes. In addition, inclusion of an enterotoxin adjuvant is associated with development of multifaceted cellular and humoral immune responses to vaccination. Here, we review exciting progress in the past few years in clinical trials for safety and efficacy, preclinical vaccines studies, and new mechanistic insights for enterotoxin adjuvants. This includes recent reports of their use in vaccines targeting microbial infections (bacterial, viral, parasitic) or substance abuse drugs.

Viral infectivity in paediatric SARS-CoV-2 clinical samples does not vary by age.

At the start of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, there was much uncertainty about the role of children in infection and transmission dynamics. Through the course of the pandemic, it became clear that children were susceptible to SARS-CoV-2 infection, although they were experiencing a notable lack of severe disease outcomes as compared to the adult population. This trend held true with the emergence of new SARS-CoV-2 variants, even in paediatric populations that were ineligible to be vaccinated. The difference in disease outcomes has prompted questions about the virological features of SARS-CoV-2 infection in this population. In order to determine if there was any difference in the infectivity of the virus produced by children with coronavirus disease 2019 (COVID-19), we compared viral RNA levels (clinical RT-qPCR C T) and infectious virus titres from 144 SARS-CoV-2-positive clinical samples collected from children aged 0 to 18 years old. We found that age had no impact on the infectiousness of SARS-CoV-2 within our cohort, with children of all ages able to produce high levels of infectious virus.

Determining the impact of vaccination on SARS-CoV-2 RT-PCR cycle threshold values and infectious viral titres.

Background: As the COVID-19 pandemic continues, efforts to better understand severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral shedding and transmission in both unvaccinated and vaccinated populations remain critical to informing public health policies and vaccine development. The utility of using real time RT-PCR cycle threshold values (CT values) as a proxy for infectious viral litres from individuals infected with SARS-CoV-2 is yet to be fully understood. This retrospective observational cohort study compares quantitative infectious viral litres derived from a focus-forming viral titre assay with SARS-CoV-2 RT-PCR CT values in both unvaccinated and vaccinated individuals infected with the Delta strain. Methods: Nasopharyngeal swabs positive for SARS-CoV-2 by RT-PCR with a CT value <27 collected from 26 June to 17 October 2021 at the University of Vermont Medical Center Clinical Laboratory for which vaccination records were available were included. Partially vaccinated and individuals <18 years of age were excluded. Infectious viral litres were determined using a micro-focus forming assay under BSL-3 containment. Results: In total, 119 specimens from 22 unvaccinated and 97 vaccinated individuals met all inclusion criteria and had sufficient residual volume to undergo viral titring. A negative correlation between RT-PCR CT values and viral litres was observed in both unvaccinated and vaccinated groups. No difference in mean CT value or viral titre was detected between vaccinated and unvaccinated groups. Viral litres did not change as a function of time since vaccination. Conclusions: Our results add to the growing body of knowledge regarding the correlation of SARS-CoV-2 RNA levels and levels of infectious virus. At similar CT values, vaccination does not appear to impact an individual's potential infectivity when infected with the Delta variant.

Intradermal fractional-dose inactivated polio vaccine (fIPV) adjuvanted with double mutant Enterotoxigenic Escherichia coli heat labile toxin (dmLT) is well-tolerated and augments a systemic immune response to all three poliovirus serotypes in a randomized placebo-controlled trial.

Eradication of poliomyelitis globally is constrained by fecal shedding of live polioviruses, both wild-type and vaccine-derived strains, into the environment. Although inactivated polio vaccines (IPV) effectively protect the recipient from clinical poliomyelitis, fecal shedding of live virus still occurs following infection with either wildtype or vaccine-derived strains of poliovirus. In the drive to eliminate the last cases of polio globally, improvements in both oral polio vaccines (OPV) (to prevent reversion to virulence) and injectable polio vaccines (to improve mucosal immunity and prevent viral shedding) are underway. The E. coli labile toxin with two or "double" attenuating mutations (dmLT) may boost immunologic responses to IPV, including at mucosal sites. We performed a double-blinded phase I controlled clinical trial to evaluate safety, tolerability, as well as systemic and mucosal immunogenicity of IPV adjuvanted with dmLT, given as a fractional (1/5th) dose intradermally (fIPV-dmLT). Twenty-nine volunteers with no past exposure to OPV were randomized to a single dose of fIPV-dmLT or fIPV alone. fIPV-dmLT was well tolerated, although three subjects had mild but persistent induration and hyperpigmentation at the injection site. A ≥ 4-fold rise in serotype-specific neutralizing antibody (SNA) titers to all three serotypes was seen in 84% of subjects receiving fIPV-dmLT vs. 50% of volunteers receiving IPV alone. SNA titers were higher in the dmLT-adjuvanted group, but only differences in serotype 1 were significant. Mucosal immune responses, as measured by polio serotype specific fecal IgA were minimal in both groups and differences were not seen. fIPV-dmLT may offer a benefit over IPV alone. Beyond NAB responses protecting the individual, studies demonstrating the ability of fIPV-dmLT to prevent viral shedding are necessary. Studies employing controlled human infection models, using monovalent OPV post-vaccine are ongoing. Studies specifically in children may also be necessary and additional biomarkers of mucosal immune responses in this population are needed. Clinicaltrials.gov Identifer: NCT03922061.

Viral infectivity in pediatric SARS-CoV-2 clinical samples does not vary by age.

During the early months of the SARS-CoV-2 pandemic, notable uncertainty emerged regarding the role of children in transmission dynamics. With time, it became more clear that children were susceptible to infection with SARS-CoV-2, but that the vast majority of children experienced mild symptoms with lower incidence of severe disease. This pattern remained consistent despite the later emergence of SARS-CoV-2 variants, including Delta and Omicron, even among children <5 ineligible for vaccination. The relative lack of severe disease in the pediatric population raised questions regarding viral kinetics and infectivity in children versus adults. We hypothesized that unique virologic features in children could explain this apparent decrease in symptoms and transmissibility early in the pandemic.

An international, interlaboratory ring trial confirms the feasibility of an extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is a transparent and accessible qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that direct RT-PCR assay methods can be clearly translated across sites utilizing readily available equipment and expertise and are thus a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Clinical Performance of Mycobacterial Immunohistochemistry in Anatomic Pathology Specimens.

OBJECTIVES: Diagnosis of mycobacterial infections poses significant challenges in anatomic pathology. We recently described the use of antimycobacteria immunohistochemistry (IHC) as a sensitive, efficient diagnostic tool and now report the clinical performance of this assay among general, noninfectious disease pathology-trained anatomic pathologists. METHODS: Over a 2-year period, all cases were retrospectively identified in which mycobacterial IHC was performed during routine diagnostic workup. RESULTS: From October 2017 to September 2019, mycobacterial IHC was evaluated for 267 cases, resulting in 58 (22%) positive stains. Compared with culture and molecular results, the sensitivity and specificity of IHC were 52% and 80%, respectively. IHC performed significantly better than acid-fast bacilli (AFB) staining (Ziehl-Neelsen) (P < .0001; sensitivity 21%, specificity 92%) but similarly to modified AFB staining (mAFB; Fite-Faraco) (P = .9; sensitivity 61%, specificity 84%). In cases with discordant IHC and mAFB staining, there were no differences in rates of culture or polymerase chain reaction-confirmed positivity. CONCLUSIONS: Mycobacterial IHC was well adopted with superior clinical performance to AFB and comparable performance to mAFB. These results support the use of IHC as an adjunctive tool in the diagnosis of mycobacterial infections and suggests its potential role as a rapid screening test for molecular testing.

Scedosporium and Lomentospora Infections Are Infrequent, Difficult to Diagnose by Histology, and Highly Virulent.

OBJECTIVE: To further characterize the histomorphology and clinicopathologic features of colonization and invasive disease by Scedosporium and Lomentospora. METHODS: We conducted a 20-year retrospective study. Patients with at least 1 histopathology specimen and concurrent culture were included. Clinical features, histopathology, microbiology, and outcomes were analyzed. RESULTS: Eighteen patients were identified, and all were immunocompromised. Eight patients had colonization, while 10 had invasive disease (pneumonia [n = 3], skin and soft-tissue infections [n = 3], disseminated disease [n = 4]). Scedosporium apiospermum was identified in 15 patients, Lomentospora prolificans in 2 patients, and Scedosporium ellipsoideum in 1 patient. Fungal elements were identified histologically in 11 patients. Granulomatous, suppurative, and necrotizing inflammation with irregular branching hyphae and characteristic microconidia were observed in 9 cases; conidiogenous cells were identified in 4 cases. Seven patients died of invasive disease despite therapy, and 3 recovered after treatment. No deaths were observed in patients with colonization. CONCLUSIONS: Scedosporium and Lomentospora are rare, virulent opportunistic fungal pathogens. Fungal morphology may overlap with other hyaline molds, but identification of obovoid conidia should allow a diagnosis of non-Aspergillus hyalohyphomycosis and consideration of Scedosporium and Lomentospora. Histopathologic correlation with culture and polymerase chain reaction is critical for diagnosis and treatment.

Dynamic Colonization of Microbes and Their Functions after Fecal Microbiota Transplantation for Inflammatory Bowel Disease.

For fecal microbiota transplantation (FMT) to be successful in immune diseases like inflammatory bowel disease, it is assumed that therapeutic microbes and their beneficial functions and immune interactions must colonize a recipient patient and persist in sufficient quantity and for a sufficient period of time to produce a clinical benefit. Few studies, however, have comprehensively profiled the colonization and persistence of transferred microbes along with the transfer of their microbial functions and interactions with the host immune system. Using 16S, metagenomic, and immunoglobulin A (IgA) sequencing, we analyzed hundreds of longitudinal microbiome samples from a randomized controlled trial of 12 patients with ulcerative colitis who received fecal transplant or placebo for 12 weeks. We uncovered diverse competitive dynamics among donor and patient strains, showing that persistence of transferred microbes is far from static. Indeed, one patient experienced a dramatic loss of donor bacteria 10 weeks into the trial, coinciding with a bloom of pathogenic bacteria and worsening symptoms. We evaluated the transfer of microbial functions, including desired ones, such as butyrate production, and unintended ones, such as antibiotic resistance. By profiling bacteria coated with IgA, we identified bacteria associated with inflammation and found that microbial interactions with the host immune system can be transferred across people, which could play a role in gut microbiome therapeutics for immune-related diseases. Our findings shed light on the colonization dynamics of gut microbes and their functions in the context of FMT to treat a complex disease-information that may provide a foundation for developing more-targeted therapeutics. IMPORTANCE Fecal microbiota transplantation (FMT)-transferring fecal microbes from a healthy donor to a sick patient-has shown promise for gut diseases such as inflammatory bowel disease. Unlike pharmaceuticals, however, fecal transplants are complex mixtures of living organisms, which must then interact with the microbes and immune system of the recipient. We sought to understand these interactions by tracking the microbes of 12 inflammatory bowel disease patients who received fecal transplants for 12 weeks. We uncovered a range of dynamics. For example, one patient experienced successful transfer of donor bacteria, only to lose them after 10 weeks. We similarly evaluated transfer of microbial functions, including how they interacted with the recipient's immune system. Our findings shed light on the colonization dynamics of gut microbes, as well as their functions in the context of FMT-information that may provide a critical foundation for the development of more-targeted therapeutics.

All Hands-On Deck and All Decks on Hand: Surmounting Supply Chain Limitations During the COVID-19 Pandemic.

Testing during the COVID-19 pandemic has been crucial to public health surveillance and clinical care. Supply chain constraints-spanning limitations in testing kits, reagents, pipet tips, and swabs availability-have challenged the ability to scale COVID-19 testing. During the early months, sample collection kits shortages constrained planned testing expansions. In response, the University of Vermont Medical Center, University of Vermont College of Medicine, Vermont Department of Health Laboratory, Aspenti Health, and providers across Vermont including 16 area hospitals partnered to surmount these barriers. The primary objectives were to increase supply availability and manage utilization. Within the first month of Vermont's stay-at-home order, the University of Vermont Medical Center laboratory partnered with College of Medicine to create in-house collection kits, producing 5000 per week. University of Vermont Medical Center reassigned 4 phlebotomists, laboratory educators, and other laboratory staff, who had reduced workloads, to participate (requiring a total of 5.3-7.6 full-time equivalent (FTE) during the period of study). By August, automation at a local commercial laboratory produced 22,000 vials of media in one week (reducing the required personnel by 1.2 FTE). A multisite, cross-institutional approach was used to manage specimen collection kit utilization across Vermont. Hospital laboratory directors, managers, and providers agreed to order only as needed to avoid supply stockpiles and supported operational constraints through ongoing validations and kit assembly. Throughout this pandemic, Vermont has ranked highly in number of tests per million people, demonstrating the value of local collaboration to surmount obstacles during disease outbreaks and the importance of creative allocation of resources to address statewide needs.

An international, interlaboratory ring trial confirms the feasibility of an open-source, extraction-less "direct" RT-qPCR method for reliable detection of SARS-CoV-2 RNA in clinical samples.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used worldwide to test and trace the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). "Extraction-less" or "direct" real time-reverse transcription polymerase chain reaction (RT-PCR) is an open-access qualitative method for SARS-CoV-2 detection from nasopharyngeal or oral pharyngeal samples with the potential to generate actionable data more quickly, at a lower cost, and with fewer experimental resources than full RT-qPCR. This study engaged 10 global testing sites, including laboratories currently experiencing testing limitations due to reagent or equipment shortages, in an international interlaboratory ring trial. Participating laboratories were provided a common protocol, common reagents, aliquots of identical pooled clinical samples, and purified nucleic acids and used their existing in-house equipment. We observed 100% concordance across laboratories in the correct identification of all positive and negative samples, with highly similar cycle threshold values. The test also performed well when applied to locally collected patient nasopharyngeal samples, provided the viral transport media did not contain charcoal or guanidine, both of which appeared to potently inhibit the RT-PCR reaction. Our results suggest that open-access, direct RT-PCR assays are a feasible option for more efficient COVID-19 coronavirus disease testing as demanded by the continuing pandemic.

Fulminant Acanthamoeba castellanii Encephalitis in an Ibrutinib-Treated Patient.

We report a case of fulminant Acanthamoeba castellanii encephalitis in a patient with chronic lymphocytic leukemia treated with ibrutinib. The unusually rapid neurologic decline and fatal outcome observed are probably related to alterations in immunologic function associated with inhibition of Bruton tyrosine kinase.