Comparison of Clostridioides difficile nucleic acid amplification test (NAAT) results using fresh and frozen stool specimens and rectal swabs.

IMPORTANCE: Nucleic acid amplification tests (NAATs) are frequently used in Clostridioides difficile research and diagnostic testing, but the effect of freezing specimens on C. difficile NAAT performance is not well characterized. This study evaluated the concordance of NAAT results between fresh and frozen specimens (fecal and rectal swabs) and found it to be very good to excellent. The results indicate that frozen fecal and rectal swab specimens may be used for C. difficile NAAT testing in research when fresh specimens are not available.

Development of a scoring system to identify high-yield specimens for bacterial broad-range 16S rRNA gene PCR with sequencing at a tertiary care medical center.

OBJECTIVES: Broad-range bacterial polymerase chain reaction with sequencing (BRBPS) provides valuable diagnostic data, especially in cases of culture-negative infections. However, as BRBPS testing demonstrates generally low positivity, cost per impactful result can be high and commonly involves longer turnaround times compared with other methods, targeting use of this assay to high-yield situations remains a challenging goal. Diagnostic stewardship can help alleviate these challenges and increase clinical utility, yet not all laboratories have a dedicated stewardship team, and little formal guidance exists on identifying high-yield samples outside of specific clinical syndromes. In this study, we performed a retrospective review of 86 BRBPS orders from a tertiary care medical center, with a focus on identifying high-yield cases using an infectious markers scoring system, visualized inflammation or organism (VIO) score, to predict return of actionable diagnostic data. METHODS: Using chart review, we evaluated how results from high VIO score or low VIO score specimens contributed to clinical management. RESULTS: Testing low VIO score samples identified an organism in only 10% of samples, and of these positive results, 33% were considered to represent contamination. Despite negative routine workup and no identified pathogen via BRBPS, broad antimicrobial treatment was continued in 85% of cases with a low VIO score. In contrast, specimens with high VIO scores were more predictably positive by BRBPS, identified organisms that were universally considered pathogens, and provided opportunities to target or de-escalate antimicrobial therapy. CONCLUSIONS: This study describes the VIO scoring system to guide the identification of high-yield samples and steward the appropriate use of BRBPS testing.

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.

Implementation of an Extraction-Free COVID Real-Time PCR Workflow in a Pediatric Hospital Setting.

BACKGROUND: This study outlines the development, implementation, and impact of a laboratory-developed, extraction-free real-time PCR assay as the primary diagnostic test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a pediatric hospital. METHODS: Clinical specimens from both upper and lower respiratory tract sources were validated, including nasopharyngeal aspirates, nasopharyngeal swabs, anterior nares swabs, and tracheal aspirates (n = 333 clinical samples). Testing volumes and laboratory turnaround times were then compared before and after implementation to investigate effects of the workflow changes. RESULTS: Compared to magnetic-bead extraction platforms, extraction-free real-time PCR demonstrated ≥95% positive agreement and ≥97% negative agreement across all tested sources. Implementation of this workflow reduced laboratory turnaround time from an average of 8.8 (+/-5.5) h to 3.6 (+/-1.3) h despite increasing testing volumes (from 1515 to 4884 tests per week over the reported period of testing). CONCLUSIONS: The extraction-free workflow reduced extraction reagent cost for SARS-CoV-2 testing by 97%, shortened sample handling time, and significantly alleviated supply chain scarcities due to the elimination of specialized extraction reagents for routine testing. Overall, this assay is a viable option for laboratories to increase efficiency and navigate reagent shortages for SARS-CoV-2 diagnostic testing.

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.

Heterogeneity of Antiviral Responses in the Upper Respiratory Tract Mediates Differential Non-lytic Clearance of Influenza Viruses.

Influenza viruses initiate infection in the upper respiratory tract (URT), but early viral tropism and the importance of cell-type-specific antiviral responses in this tissue remain incompletely understood. By infecting transgenic lox-stop-lox reporter mice with a Cre-recombinase-expressing influenza B virus, we identify olfactory sensory neurons (OSNs) as a major viral cell target in the URT. These cells become infected, then eliminate the virus and survive in the host post-resolution of infection. OSN responses to infection are characterized by a strong induction of interferon-stimulated genes and more rapid clearance of viral protein relative to other cells in the epithelium. We speculate that this cell-type-specific response likely serves to protect the central nervous system from infection. More broadly, these results highlight the importance of evaluating antiviral responses across different cell types, even those within the same tissue, to more fully understand the mechanisms of viral disease.

Long-term surviving influenza infected cells evade CD8+ T cell mediated clearance.

Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects multiple epithelial cell subsets in the respiratory tract, eliciting damage to the lungs. Clearance of IAV is primarily dependent on CD8+ T cells, which must balance control of the infection with immunopathology. Using a virus expressing Cre recombinase to permanently label infected cells in a Cre-inducible reporter mouse, we previously discovered infected club cells that survive both lytic virus replication and CD8+ T cell-mediated clearance. In this study, we demonstrate that ciliated epithelial cells, type I and type II alveolar cells can also become survivor cells. Survivor cells are stable in the lung long-term and demonstrate enhanced proliferation compared to uninfected cells. When we investigated how survivor cells evade CD8+ T cell killing we observed that survivor cells upregulated the inhibitory ligand PD-L1, but survivor cells did not use PD-L1 to evade CD8+ T cell killing. Instead our data suggest that survivor cells are not inherently resistant to CD8+ T cell killing, but instead no longer present IAV antigen and cannot be detected by CD8+ T cells. Finally, we evaluate the failure of CD8+ T cells to kill these previously infected cells. This work demonstrates that additional cell types can survive IAV infection and that these cells robustly proliferate and are stable long term. By sparing previously infected cells, the adaptive immune system may be minimizing pathology associated with IAV infection.

The Development and Use of Reporter Influenza B Viruses.

Influenza B viruses (IBVs) are major contributors to total human influenza disease, responsible for ~1/3 of all infections. These viruses, however, are relatively less studied than the related influenza A viruses (IAVs). While it has historically been assumed that the viral biology and mechanisms of pathogenesis for all influenza viruses were highly similar, studies have shown that IBVs possess unique characteristics. Relative to IAV, IBV encodes distinct viral proteins, displays a different mutational rate, has unique patterns of tropism, and elicits different immune responses. More work is therefore required to define the mechanisms of IBV pathogenesis. One valuable approach to characterize mechanisms of microbial disease is the use of genetically modified pathogens that harbor exogenous reporter genes. Over the last few years, IBV reporter viruses have been developed and used to provide new insights into the host response to infection, viral spread, and the testing of antiviral therapeutics. In this review, we will highlight the history and study of IBVs with particular emphasis on the use of genetically modified viruses and discuss some remaining gaps in knowledge that can be addressed using reporter expressing IBVs.

DNA mismatch repair is required for the host innate response and controls cellular fate after influenza virus infection.

Despite the cytopathic nature of influenza A virus (IAV) replication, we recently reported that a subset of lung epithelial club cells is able to intrinsically clear the virus and survive infection. However, the mechanisms that drive cell survival during a normally lytic infection remained unclear. Using a loss-of-function screening approach, we discovered that the DNA mismatch repair (MMR) pathway is essential for club cell survival of IAV infection. Repair of virally induced oxidative damage by the DNA MMR pathway not only allowed cell survival of infection, but also facilitated host gene transcription, including the expression of antiviral and stress response genes. Enhanced viral suppression of the DNA MMR pathway prevented club cell survival and increased the severity of viral disease in vivo. Altogether, these results identify previously unappreciated roles for DNA MMR as a central modulator of cellular fate and a contributor to the innate antiviral response, which together control influenza viral disease severity.

Non-lytic clearance of influenza B virus from infected cells preserves epithelial barrier function.

Influenza B virus (IBV) is an acute, respiratory RNA virus that has been assumed to induce the eventual death of all infected cells. We and others have shown however, that infection with apparently cytopathic viruses does not necessarily lead to cell death; some cells can intrinsically clear the virus and persist in the host long-term. To determine if any cells can survive direct IBV infection, we here generate a recombinant IBV capable of activating a host-cell reporter to permanently label all infected cells. Using this system, we demonstrate that IBV infection leads to the formation of a survivor cell population in the proximal airways that are ciliated-like, but transcriptionally and phenotypically distinct from both actively infected and bystander ciliated cells. We also show that survivor cells are critical to maintain respiratory barrier function. These results highlight a host response pathway that preserves the epithelium to limit the severity of IBV disease.

Epitranscriptomic Enhancement of Influenza A Virus Gene Expression and Replication.

Many viral RNAs are modified by methylation of the N6 position of adenosine (m6A). m6A is thought to regulate RNA splicing, stability, translation, and secondary structure. Influenza A virus (IAV) expresses m6A-modified RNAs, but the effects of m6A on this segmented RNA virus remain unclear. We demonstrate that global inhibition of m6A addition inhibits IAV gene expression and replication. In contrast, overexpression of the cellular m6A "reader" protein YTHDF2 increases IAV gene expression and replication. To address whether m6A residues modulate IAV RNA function in cis, we mapped m6A residues on the IAV plus (mRNA) and minus (vRNA) strands and used synonymous mutations to ablate m6A on both strands of the hemagglutinin (HA) segment. These mutations inhibited HA mRNA and protein expression while leaving other IAV mRNAs and proteins unaffected, and they also resulted in reduced IAV pathogenicity in mice. Thus, m6A residues in IAV transcripts enhance viral gene expression.

A CRISPR Activation Screen Identifies a Pan-avian Influenza Virus Inhibitory Host Factor.

Influenza A virus (IAV) is a pathogen that poses significant risks to human health. It is therefore critical to develop strategies to prevent influenza disease. Many loss-of-function screens have been performed to identify the host proteins required for viral infection. However, there has been no systematic screen to identify the host factors that, when overexpressed, are sufficient to prevent infection. In this study, we used CRISPR/dCas9 activation technology to perform a genome-wide overexpression screen to identify IAV restriction factors. The major hit from our screen, B4GALNT2, showed inhibitory activity against influenza viruses with an α2,3-linked sialic acid receptor preference. B4GALNT2 overexpression prevented the infection of every avian influenza virus strain tested, including the H5, H9, and H7 subtypes, which have previously caused disease in humans. Thus, we have used CRISPR/dCas9 activation technology to identify a factor that can abolish infection by avian influenza viruses.

Rationally Designed Influenza Virus Vaccines That Are Antigenically Stable during Growth in Eggs.

Influenza virus vaccine production is currently limited by the ability to grow circulating human strains in chicken eggs or in cell culture. To facilitate cost-effective growth, vaccine strains are serially passaged under production conditions, which frequently results in mutations of the major antigenic protein, the viral hemagglutinin (HA). Human vaccination with an antigenically drifted strain is known to contribute to poor vaccine efficacy. To address this problem, we developed a replication-competent influenza A virus (IAV) with an artificial genomic organization that allowed the incorporation of two independent and functional HA proteins with different growth requirements onto the same virion. Vaccination with these viruses induced protective immunity against both strains from which the HA proteins were derived, and the magnitude of the response was as high as or higher than vaccination with either of the monovalent parental strains alone. Dual-HA viruses also displayed remarkable antigenic stability; even when using an HA protein known to be highly unstable during growth in eggs, we observed high-titer virus amplification without a single adaptive mutation. Thus, the viral genomic design described in this work can be used to grow influenza virus vaccines to high titers without introducing antigenic mutations.IMPORTANCE Influenza A virus (IAV) is a major public health threat, and vaccination is currently the best available strategy to prevent infection. While there have been many advances in influenza vaccine production, the fact that we cannot predict the growth characteristics of a given strain under vaccine production conditions a priori introduces fundamental uncertainty into the process. Clinically relevant IAV strains frequently grow poorly under vaccine conditions, and this poor growth can result in the delay of vaccine production or the exchange of the recommended strain for one with favorable growth properties. Even in strains that grow to high titers, adaptive mutations in the antigenic protein hemagglutinin (HA) that make it antigenically dissimilar to the circulating strain are common. The genomic restructuring of the influenza virus described in this work offers a solution to the problem of uncertain or unstable growth of IAV during vaccine production.

Thiol-ene Photocrosslinking of Cytocompatible Resilin-Like Polypeptide-PEG Hydrogels.

A range of chemical strategies have been used for crosslinking recombinant polypeptide hydrogels, although only a few have employed photocrosslinking approaches. Here, we capitalize on the novel insect protein, resilin, and the versatility of click reactions to introduce a resilin-like polypeptide (RLP) that is capable of photoinitiated thiol-ene crosslinking. Lysine residues of the RLP were functionalized with norbornene acid as confirmed via 1H-NMR spectroscopy. The RLPNs were subsequently photocrosslinked with multi-arm PEG thiols in the presence of a photoinitiator to form elastic hybrid hydrogels. The crosslinking reaction and resulting RLP-PEG networks demonstrated cytocompatibility with human mesenchymal stem cells in both 2D cell-adhesion and 3D photoencapsulation studies.