Case report: isolation of Hydrogenophaga from septic blood culture following near-death drowning in lakewater.

A patient suffered a non-fatal wet drowning in a freshwater lake and developed bacteraemia several days later. Blood culture grew a Gram-negative rod that could not be identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 16S ribosomal RNA sequencing of the isolate identified the microbe as Hydrogenophaga laconesensis - an environmental microbe commonly found in freshwater. The recovery of multiple pathogenic micro-organisms (although not H. laconesensis ) from culture of respiratory specimens prompted the initiation of antibiotic therapy with cefepime and, later, vancomycin. The patient's clinical course gradually improved over the course of 2 weeks and she was ultimately discharged home with minimal sequelae. To our knowledge, this is the first evidence of human infection with bacteria in the genus Hydrogenophaga . Hydrogenophaga may be considered in cases of freshwater near-drowning, and MALDI-TOF MS databases should be updated to include H. laconesensis .

Development of a rapid, simple, and sensitive point-of-care technology platform utilizing ternary NanoLuc.

Point-of-care tests are highly valuable in providing fast results for medical decisions for greater flexibility in patient care. Many diagnostic tests, such as ELISAs, that are commonly used within clinical laboratory settings require trained technicians, laborious workflows, and complex instrumentation hindering their translation into point-of-care applications. Herein, we demonstrate the use of a homogeneous, bioluminescent-based, split reporter platform that enables a simple, sensitive, and rapid method for analyte detection in clinical samples. We developed this point-of-care application using an optimized ternary, split-NanoLuc luciferase reporter system that consists of two small reporter peptides added as appendages to analyte-specific affinity reagents. A bright, stable bioluminescent signal is generated as the affinity reagents bind to the analyte, allowing for proximity-induced complementation between the two reporter peptides and the polypeptide protein, in addition to the furimazine substrate. Through lyophilization of the stabilized reporter system with the formulated substrate, we demonstrate a shelf-stable, all-in-one, add-and-read analyte-detection system for use in complex sample matrices at the point-of-care. We highlight the modularity of this platform using two distinct SARS-CoV-2 model systems: SARS-CoV-2 N-antigen detection for active infections and anti-SARS-CoV-2 antibodies for immunity status detection using chemically conjugated or genetically fused affinity reagents, respectively. This technology provides a simple and standardized method to develop rapid, robust, and sensitive analyte-detection assays with flexible assay formatting making this an ideal platform for research, clinical laboratory, as well as point-of-care applications utilizing a simple handheld luminometer.

SARS-CoV-2 and other respiratory pathogens are detected in continuous air samples from congregate settings.

Two years after the emergence of SARS-CoV-2, there is still a need for better ways to assess the risk of transmission in congregate spaces. We deployed active air samplers to monitor the presence of SARS-CoV-2 in real-world settings across communities in the Upper Midwestern states of Wisconsin and Minnesota. Over 29 weeks, we collected 527 air samples from 15 congregate settings. We detected 106 samples that were positive for SARS-CoV-2 viral RNA, demonstrating that SARS-CoV-2 can be detected in continuous air samples collected from a variety of real-world settings. We expanded the utility of air surveillance to test for 40 other respiratory pathogens. Surveillance data revealed differences in timing and location of SARS-CoV-2 and influenza A virus detection. In addition, we obtained SARS-CoV-2 genome sequences from air samples to identify variant lineages. Collectively, this shows air sampling is a scalable, high throughput surveillance tool that could be used in conjunction with other methods for detecting respiratory pathogens in congregate settings.

Characterization of the SARS-CoV-2 B.1.621 (Mu) variant.

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, Beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4- to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently rechallenged with the B.1.621 variant, suggesting that the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

SARS-CoV-2 and other respiratory pathogens are detected in continuous air samples from congregate settings.

Two years after the emergence of SARS-CoV-2, there is still a need for better ways to assess the risk of transmission in congregate spaces. We deployed active air samplers to monitor the presence of SARS-CoV-2 in real-world settings across communities in the Upper Midwestern states of Wisconsin and Minnesota. Over 29 weeks, we collected 527 air samples from 15 congregate settings and detected 106 SARS-CoV-2 positive samples, demonstrating SARS-CoV-2 can be detected in air collected from daily and weekly sampling intervals. We expanded the utility of air surveillance to test for 40 other respiratory pathogens. Surveillance data revealed differences in timing and location of SARS-CoV-2 and influenza A virus detection in the community. In addition, we obtained SARS-CoV-2 genome sequences from air samples to identify variant lineages. Collectively, this shows air surveillance is a scalable, cost-effective, and high throughput alternative to individual testing for detecting respiratory pathogens in congregate settings.

A "Null" Pattern of p16 Immunostaining in Endometrial Serous Carcinoma: An Under-recognized and Important Aberrant Staining Pattern.

The ability to distinguish endometrial serous carcinoma (SC) from high-grade endometrioid adenocarcinoma is of great importance given their differences in prognosis and management. In practice, this distinction typically relies upon the use of a focused immunohistochemical panel including p53, p16, and mismatch repair proteins. The expression of p16 is characteristically strong and diffuse in SCs, and weak and/or patchy in many high-grade endometrioid adenocarcinomas. Here, we report a subset of SCs that are entirely negative for p16 immunostaining, a pattern we refer to as "p16 null." This pattern was identified in 2 of 63 cases of SC diagnosed at our institution-1 with histologically classic features and 1 with ambiguous high-grade histologic features. These tumors otherwise showed a SC signature by immunohistochemical and demonstrated an SC pattern of genetic mutations. No mutation in the gene for p16, cyclin-dependent kinase inhibitor 2A (CDKN2A), was identified in either case. However, molecular correlates for the absent p16 expression were present, including homozygous deletion of CDKN2A in one case and hemizygous deletion of CDKN2A with promotor hypermethylation of the remaining allele in the other case. To our knowledge, this constitutes the first report conclusively demonstrating the existence of a small subset of SCs that are completely negative by p16 immunohistochemistry, and the molecular lesions responsible for this pattern. In the context of an otherwise clinically and histologically classic example of SC, we endorse this "null" p16 staining pattern as an alternative aberrant staining pattern that should not deter one from committing to this diagnosis.

Acute SARS-CoV-2 infections harbor limited within-host diversity and transmit via tight transmission bottlenecks.

The emergence of divergent SARS-CoV-2 lineages has raised concern that novel variants eliciting immune escape or the ability to displace circulating lineages could emerge within individual hosts. Though growing evidence suggests that novel variants arise during prolonged infections, most infections are acute. Understanding how efficiently variants emerge and transmit among acutely-infected hosts is therefore critical for predicting the pace of long-term SARS-CoV-2 evolution. To characterize how within-host diversity is generated and propagated, we combine extensive laboratory and bioinformatic controls with metrics of within- and between-host diversity to 133 SARS-CoV-2 genomes from acutely-infected individuals. We find that within-host diversity is low and transmission bottlenecks are narrow, with very few viruses founding most infections. Within-host variants are rarely transmitted, even among individuals within the same household, and are rarely detected along phylogenetically linked infections in the broader community. These findings suggest that most variation generated within-host is lost during transmission.

Oil immersed lossless total analysis system for integrated RNA extraction and detection of SARS-CoV-2.

The COVID-19 pandemic exposed difficulties in scaling current quantitative PCR (qPCR)-based diagnostic methodologies for large-scale infectious disease testing. Bottlenecks include lengthy multi-step processes for nucleic acid extraction followed by qPCR readouts, which require costly instrumentation and infrastructure, as well as reagent and plastic consumable shortages stemming from supply chain constraints. Here we report an Oil Immersed Lossless Total Analysis System (OIL-TAS), which integrates RNA extraction and detection onto a single device that is simple, rapid, cost effective, and requires minimal supplies and infrastructure to perform. We validated the performance of OIL-TAS using contrived SARS-CoV-2 viral particle samples and clinical nasopharyngeal swab samples. OIL-TAS showed a 93% positive predictive agreement (n = 57) and 100% negative predictive agreement (n = 10) with clinical SARS-CoV-2 qPCR assays in testing clinical samples, highlighting its potential to be a faster, cheaper, and easier-to-deploy alternative for infectious disease testing.

Viral Sequencing to Investigate Sources of SARS-CoV-2 Infection in US Healthcare Personnel.

BACKGROUND: Healthcare personnel (HCP) are at increased risk of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We posit that current infection control guidelines generally protect HCP from SARS-CoV-2 infection in a healthcare setting. METHODS: In this retrospective case series, we used viral genomics to investigate the likely source of SARS-CoV-2 infection in HCP at a major academic medical institution in the Upper Midwest of the United States between 25 March and 27 December 2020. We obtained limited epidemiological data through informal interviews and review of the electronic health record and combined this information with healthcare-associated viral sequences and viral sequences collected in the broader community to infer the most likely source of infection in HCP. RESULTS: We investigated SARS-CoV-2 infection clusters involving 95 HCP and 137 possible patient contact sequences. The majority of HCP infections could not be linked to a patient or coworker (55 of 95 [57.9%]) and were genetically similar to viruses circulating concurrently in the community. We found that 10.5% of HCP infections (10 of 95) could be traced to a coworker. Strikingly, only 4.2% (4 of 95) could be traced to a patient source. CONCLUSIONS: Infections among HCP add further strain to the healthcare system and put patients, HCP, and communities at risk. We found no evidence for healthcare-associated transmission in the majority of HCP infections evaluated. Although we cannot rule out the possibility of cryptic healthcare-associated transmission, it appears that HCP most commonly become infected with SARS-CoV-2 via community exposure. This emphasizes the ongoing importance of mask wearing, physical distancing, robust testing programs, and rapid distribution of vaccines.

Initial evaluation of a mobile SARS-CoV-2 RT-LAMP testing strategy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16 to November 19, 2020, 4,704 surveillance samples were collected from volunteers and tested for SARS-CoV-2 at 5 sites. A total of 21 samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, while 8 were negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the RT-LAMP assay's false-negative rate from July 16 to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or less and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP negative pools (2,493 samples) testing positive in the more sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

Fully automated detection and differentiation of pandemic and endemic coronaviruses (NL63, 229E, HKU1, OC43 and SARS-CoV-2) on the hologic panther fusion.

The hologic panther fusion (PF) platform provides fully automated CE marked diagnostics for respiratory viruses, including the recently discovered severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) by a transcription mediated amplification (TMA) assay, but not for the endemic human coronaviruses (hCoV). Therefore, a laboratory developed test (LDT) comprising a multiplexed reverse transcription polymerase chain reaction (RT-PCR) protocol that detects and differentiates the four hCoV NL63, 229E, HKU1, and OC43 was adapted on the PF. The novel CE marked Aptima SARS-CoV-2 TMA and the LDT for hCoV were validated with 321 diagnostic specimens from the upper and lower respiratory tract in comparison to two SARS-CoV-2 RT-PCRs (PF E-gene RT-PCR and genesig RT-PCR, 157 specimens) or the R-GENE hCoV/hParaFlu RT-PCR (164 specimens), respectively. For the endemic hCoV, results were 96.3% concordant with two specimens discordantly positive in the PF and four specimens discordantly positive in the R-GENE assay. All discordantly positive samples had Ct values between 33 and 39. The PF hCoV LDT identified 23 hCoV positive specimens as NL63, 15 as 229E, 15 as HKU1, and 25 as OC43. The Aptima SARS-CoV-2 TMA gave 99.4% concordant results compared to the consensus results with a single specimen discordantly positive. Moreover, 36 samples from proficiency testing panels were detected and typed correctly by both novel methods. In conclusion, the SARS-CoV-2 TMA and the LDT for hCoV enhanced the diagnostic spectrum of the PF for all coronaviruses circulating globally for a multitude of diagnostic materials from the upper and lower respiratory tract.

Initial Evaluation of a Mobile SARS-CoV-2 RT-LAMP Testing Strategy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) control in the United States remains hampered, in part, by testing limitations. We evaluated a simple, outdoor, mobile, colorimetric reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay workflow where self-collected saliva is tested for SARS-CoV-2 RNA. From July 16, 2020, to November 19, 2020, surveillance samples (n = 4704) were collected from volunteers and tested for SARS-CoV-2 at 5 sites. Twenty-one samples tested positive for SARS-CoV-2 by RT-LAMP; 12 were confirmed positive by subsequent quantitative reverse-transcription polymerase chain reaction (qRT-PCR) testing, whereas 8 tested negative for SARS-CoV-2 RNA, and 1 could not be confirmed because the donor did not consent to further molecular testing. We estimated the false-negative rate of the RT-LAMP assay only from July 16, 2020, to September 17, 2020 by pooling residual heat-inactivated saliva that was unambiguously negative by RT-LAMP into groups of 6 or fewer and testing for SARS-CoV-2 RNA by qRT-PCR. We observed a 98.8% concordance between the RT-LAMP and qRT-PCR assays, with only 5 of 421 RT-LAMP-negative pools (2493 total samples) testing positive in the more-sensitive qRT-PCR assay. Overall, we demonstrate a rapid testing method that can be implemented outside the traditional laboratory setting by individuals with basic molecular biology skills and that can effectively identify asymptomatic individuals who would not typically meet the criteria for symptom-based testing modalities.

Optimizing direct RT-LAMP to detect transmissible SARS-CoV-2 from primary nasopharyngeal swab samples.

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/µl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.

Revealing fine-scale spatiotemporal differences in SARS-CoV-2 introduction and spread.

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties following the statewide "Safer at Home" order, which went into effect 25 March 2020. Our results suggest patterns of SARS-CoV-2 transmission may vary substantially even in nearby communities. Understanding these local patterns will enable better targeting of public health interventions.

Distinct patterns of SARS-CoV-2 transmission in two nearby communities in Wisconsin, USA.

Evidence-based public health approaches that minimize the introduction and spread of new SARS-CoV-2 transmission clusters are urgently needed in the United States and other countries struggling with expanding epidemics. Here we analyze 247 full-genome SARS-CoV-2 sequences from two nearby communities in Wisconsin, USA, and find surprisingly distinct patterns of viral spread. Dane County had the 12th known introduction of SARS-CoV-2 in the United States, but this did not lead to descendant community spread. Instead, the Dane County outbreak was seeded by multiple later introductions, followed by limited community spread. In contrast, relatively few introductions in Milwaukee County led to extensive community spread. We present evidence for reduced viral spread in both counties, and limited viral transmission between counties, following the statewide Safer-at-Home public health order, which went into effect 25 March 2020. Our results suggest that early containment efforts suppressed the spread of SARS-CoV-2 within Wisconsin.

Fatal Myocarditis Following Treatment with the PD-1 Inhibitor Nivolumab.

Therapeutic antibodies targeting the programmed cell death protein 1 (PD-1) pathway function as immune checkpoint inhibitors, allowing the immune system to recognize tumors which otherwise escape immune surveillance. However, these agents can also elicit an autoimmune response by inhibiting the ability of non-neoplastic tissues and regulatory cells to suppress the immune system. Here we present a fatal case of active myocarditis in a 55-year-old man with non-small-cell lung cancer which occurred following monotherapy with the PD-1 inhibitor nivolumab (Opdivo). He presented with acute right-sided heart failure and died 1 day after admission. Postmortem examination revealed multiple gelatinous lesions in the myocardium of the interventricular septum and the bilateral atria and ventricles which had microscopic features diagnostic of myocarditis. Subsequent studies failed to identify an infectious cause. Immune checkpoint inhibitors are an increasingly common addition to anticancer regimens and they should be considered in the evaluation of acute myocarditis.

High-risk human papillomavirus DNA detected in primary squamous cell carcinoma of urinary bladder.

CONTEXT: We reported previously that more than one-third (37%) of primary bladder squamous cell carcinomas (SCCs) demonstrate diffuse p16 immunoreactivity independent of gender. This observation made us question whether p16 overexpression in bladder carcinoma is due to human papillomavirus (HPV)-dependent mechanisms. OBJECTIVES: To determine whether the presence of high-risk HPV (HR-HPV) DNA could be detected in these tumor cells. DESIGN: Fourteen cases of primary bladder SCC, which were positive for p16 by immunohistochemistry, were probed for the detection of HR-HPV by in situ hybridization and the signal amplification Invader assay. Samples positive for detection of HR-HPV by Invader assay were amplified by using HR-HPV type-specific primers, and amplification products were DNA sequenced. RESULTS: Detection of HR-HPV by the in situ hybridization method was negative in all cases (0 of 14). However, in 3 of 14 cases (21.4%), the presence of HR-HPV DNA was detected with the Cervista HPV HR Invader assay, which was followed by identification of genotype. All positive cases were confirmed by using HR-HPV type-specific amplification followed by DNA sequencing. Identified HR-HPV genotypes included HPV 16 (2 cases) and HPV 35 (1 case). CONCLUSIONS: High-risk HPV DNA is detectable in a subset of primary bladder SCCs. Based on the well-documented carcinogenic potential of HR-HPV, there is a necessity for additional studies to investigate the role of HR-HPV in bladder carcinogenesis.

Naturally occurring genotype 2b/1a hepatitis C virus in the United States.

BACKGROUND: Hepatitis C Virus (HCV) infected patients are frequently repeatedly exposed to the virus, but very few recombinants between two genotypes have been reported. FINDINGS: We describe the discovery of an HCV recombinant using a method developed in a United States clinical lab for HCV genotyping that employs sequencing of both 5' and 3' portions of the HCV genome. Over twelve months, 133 consecutive isolates were analyzed, and a virus from one patient was found with discordant 5' and 3' sequences suggesting it was a genotype 2b/1a recombinant. We ruled out a mixed infection and mapped a recombination point near the NS2/3 cleavage site. CONCLUSIONS: This unique HCV recombinant virus described shares some features with other recombinant viruses although it is the only reported recombinant of a genotype 2 with a subtype 1a. This recombinant represents a conundrum for current clinical treatment guidelines, including treatment with protease inhibitors. This recombinant is also challenging to detect by the most commonly employed methods of genotyping that are directed primarily at the 5' structural portion of the HCV genome.

The antiviral dynamin family member, MxA, tubulates lipids and localizes to the smooth endoplasmic reticulum.

Mx proteins are induced by type I interferon and inhibit a broad range of viruses by undefined mechanisms. They are included within the dynamin family of large GTPases, which are involved in vesicle trafficking and share common biophysical features. These properties include the propensity to self-assemble, an affinity for lipids, and the ability to tubulate membranes. In this report we establish that human MxA, despite sharing only 30% homology with conventional dynamin, possesses many of these properties. We demonstrate for the first time that MxA self-assembles into rings that tubulate lipids in vitro, and associates with a specific membrane compartment in cells, the smooth endoplasmic reticulum.