Sleeping Beauty kit sets provide rapid and accessible generation of artificial antigen-presenting cells for natural killer cell expansion.

Artificial antigen-presenting cells (aAPCs) offer a cost effective and convenient tool for the expansion of chimeric antigen receptor (CAR)-bearing T cells and NK cells. aAPCs are particularly useful because of their ability to efficiently expand low-frequency antigen-reactive lymphocytes in bulk cultures. Commonly derived from the leukemic cell line K562, these aAPCs lack most major histocompatibility complex expression and are therefore useful for NK cell expansion without triggering allogeneic T-cell proliferation. To combat difficulties in accessing existing aAPC lines, while circumventing the iterative lentiviral gene transfers with antibody-mediated sorting required for the isolation of stable aAPC clones, we developed a single-step technique using Sleeping Beauty (SB)-based vectors with antibiotic selection options. Our SB vectors contain options of two to three genes encoding costimulatory molecules, membrane-bound cytokines as well as the presence of antibiotic-resistance genes that allow for stable transposition-based transfection of feeder cells. Transfection of K562 with SB vectors described in this study allows for the surface expression of CD86, 4-1BBL, membrane-bound (mb) interleukin (IL)-15 and mbIL-21 after simultaneous transposition and antibiotic selection using only two antibiotics. aAPCs successfully expanded NK cells to high purity (80-95%). Expanded NK cells could be further engineered by lentiviral CAR transduction. The multivector kit set is publicly available and will allow convenient and reproducible in-house production of effective aAPCs for the in vitro expansion of primary cells.

MicroRNA-mediated metabolic reprogramming of chimeric antigen receptor T cells.

Advances made in chimeric antigen receptor (CAR) T cell therapy have revolutionized the treatment and management of certain cancers. Currently, B cell malignancies have been among the few cancers to which CAR T cells have shown persistent and resilient anti-tumor responses. A growing body of evidence suggests that the persistence of CAR T cells within patients following infusion is linked to the mitochondrial fitness of the CAR T cell, which could affect clinical outcomes. Analysis of CAR T cells from patients undergoing successful treatment has shown an increase in mitochondrial mass and fusion events, and a reduction in aerobic metabolism, highlighting the importance of mitochondria in CAR T cell function. Consequently, there has been recent interest and investment in approaches that focus on mitochondrial programming. In this regard, miRNAs are promising agents in mitochondrial reprogramming for several reasons: (1) natural and artificial miRNAs are non-immunogenic, (2) one miRNA can simultaneously modulate the expression of multiple genes within a pathway, (3) the small size of a sequence required for producing mature miRNA is ideal for use in viral vectors and (4) different precursor miRNAs (pre-miRNAs) hairpins can be incorporated into a polycistronic miRNA cluster to create a miRNA cocktail. In this perspective, we describe the latest genetic engineering strategies that can be used to achieve the optimal expression of candidate miRNAs alongside a CAR construct. In addition, we include an in silico analysis of rational candidate miRNAs that could promote the mitochondrial fitness of CAR T cells.

Implications of SARS-CoV-2 Mutations for Genomic RNA Structure and Host microRNA Targeting.

The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.

Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon.

Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.

Chimeric antigen receptor T cell persistence and memory cell formation.

It is now becoming clear that less differentiated naive and memory T cells are superior to effector T cells in the transfer of immunity for adoptive cell therapy. This review will outline the challenges faced by chimeric antigen receptor (CAR) T cell therapy in the generation of persistence and memory for CAR T cells, and summarize recent strategies to improve CAR T cell persistence, with a focus on memory cell formation. The relevance of enhancing persistence in more differentiated effector T cells is also covered, because genetic and pharmacological interventions may prolong effector T cell activity and lifespan, thereby improving anti-cancer activity. In particular, it may be possible to enforce epigenetic changes in differentiated T cells to enhance memory CAR T cell formation. Optimizing the generation of self-renewing T cell populations (e.g. memory cells), while maintaining differentiated effector T cells through epigenome modification, will help overcome barriers to T cell expansion and survival, thereby improving clinical outcomes in CAR T cell therapy.

Role of Lymphocyte Subsets in the Immune Response to Primary B Cell-Derived Exosomes.

Exosomes are lipid nanovesicles released after fusion of the endosomal limiting membrane with the plasma membrane. In this study, we investigated the requirement for CD4 T cells, B cells, and NK cells to provide help for CD8 T cell-mediated response to B cell-derived exosomes. CTL responses to Ag-loaded exosomes were dependent on host MHC class I, with a critical role for splenic langerin+ CD8α+ dendritic cells (DCs) in exosomal Ag cross-presentation. In addition, there was an absolute dependence on the presence of CD4 T cells, CD8 T cells, and NK cells, where the loss of any one of these subsets led to a complete loss of CTL response. Interestingly, NK cell depletion experiments demonstrated a critical cutoff point for depletion efficacy, with low-level residual NK cells providing sufficient help to allow optimal CD8 T cell proliferative responses to exosomal protein. Despite the potential role for B cells in the response to B cell-derived exosomal proteins, B cell depletion did not alter the exosome-induced CTL response. Similarly, a possible role for the BCR or circulating Ab in mediating CTL responses to B cell-derived exosomes was ruled out using DHLMP2A mice, which lack secreted and membrane-bound Ab, yet harbor marginal zone and follicular B cells. In contrast, CTL responses to DC-derived exosomes were significantly inhibited within Ab-deficient DHLMP2A mice compared with wild-type mice. However, this response was not restored upon serum transfer, implicating a role for the BCR, but not circulating Ab, in DC-derived exosome responses.

Apoptotic vesicles: deathly players in cancer-associated coagulation.

Although cancer is associated with coagulation disorders, it is still unclear how the combination of tumor cell and host factors enhance the hypercoagulable state of cancer patients. Emerging evidence points to a central role for tumor endosomal and plasma membrane-derived vesicular components in the pathogenesis of cancer-related thrombosis. In particular, tumor cell membranes and extracellular vesicles (EV) harbor lipids and proteinaceous coagulation factors able to initiate multiple points within the coagulation matrix. The impact of chemotherapy upon a host already burdened with a hypercoagulable state increases the risk of pathological coagulation. We argue that chemotherapy-induced EV harbor the most active components for cancer related thrombosis and discuss how membrane components of the host and tumor act to initiate coagulation to enhance thrombotic risk in cancer patients.

Melanoma growth and lymph node metastasis is independent of host CD169 expression.

Metastasis to the lymph node is a frequent and early event in tumour dissemination. Tumour soluble factors, including extracellular vesicles, condition host organs for metastatic tumour spread, thereby facilitating tumour cell migration and survival. In the peripheral lymphatics, extracellular vesicles are captured via their sialic acids by lymph node macrophages expressing the CD169 (sialoadhesin) molecule, thereby suppressing the immune response. We hypothesised that the CD169 molecule could modulate primary tumour growth and invasion into the regional lymph node by altering the immune response to tumour extracellular vesicles, or by directly interacting with invading tumour cells. No significant difference was noted in primary tumour growth between wild-type and CD169-/- mice, and protection against tumour challenge with tumour extracellular vesicle immunisation was similar between the strains. Subcutaneous implantation of B16 (F1 or F10) into the ventral-carpal aspect of forelimb resulted in melanoma infiltration into the axillary and brachial lymph nodes. CD169-/- mice displayed a lower level of metastatic lymph node lesions, however this failed to reach statistical significance. Although CD169 participates in the immune response to tumour antigen and appears to be a positive prognostic marker for human cancers, its role in modulating melanoma growth and metastasis is less clear.

Mechanistic insight into the procoagulant activity of tumor-derived apoptotic vesicles.

BACKGROUND: Chemotherapy induces the release of apoptotic vesicles (ApoV) from the tumor plasma membrane. Tumor ApoV may enhance the risk of thrombotic events in cancer patients undergoing chemotherapy. However, the relative contribution of ApoV to coagulation and the pathways involved remain poorly characterized. In addition, this study sets out to compare the procoagulant activity of chemotherapy-induced ApoV with their cell of origin and to determine the mechanisms of ApoV-induced coagulation. METHODS: We utilized human and murine cancer cell lines and chemotherapeutic agents to determine the requirement for the coagulation factors (tissue factor; TF, FII, FV, FVII, FVIII, FIX and phosphatidylserine) in the procoagulant activity of ApoV. The role of previously identified ApoV-associated FV was determined in a FV functional assay. RESULTS: ApoV were significantly more procoagulant per microgram of protein compared to parental living or dying tumor cells. In the phase to peak fibrin generation, procoagulant activity was dependent on phosphatidylserine, TF expression, FVII and the prothrombinase complex. However, the intrinsic coagulation factors FIX and FVIII were dispensable. ApoV-associated FV could not support coagulation in the absence of supplied, exogenous FV. CONCLUSIONS: ApoV are significantly more procoagulant than their parental tumor cells. ApoV require the extrinsic tenase and prothrombinase complex to activate the early phase of coagulation. Endogenous FV identified on tumor ApoV is serum-derived and functional, but is non-essential for ApoV-mediated fibrin generation. GENERAL SIGNIFICANCE: This study clarifies the mechanisms of procoagulant activity of vesicles released from dying tumor cells.

The CD169 sialoadhesin molecule mediates cytotoxic T-cell responses to tumour apoptotic vesicles.

Apoptosis leads to the fragmentation and packaging of cellular contents into discrete vesicles, a process known as 'blebbing'. Extracellular vesicles express membrane-bound sialic acids, which enable their capture by CD169 (sialoadhesin; Siglec-1) expressing macrophages in the lymph node and spleen. Furthermore, CD169 mediates vesicle trafficking and suppresses the immune response to exosomes-a type of extracellular vesicle released from living cells. In this study, we found that CD169(+) macrophages were the predominant splenic macrophage subset responsible for the capture of EL4 lymphoma-derived apoptotic vesicles (ApoVs) from circulation. CD169(-/-) mice had significantly enhanced in vivo cytotoxic T lymphocyte responses to antigen-pulsed ApoVs, indicating a suppressive role for CD169(+) macrophages to ApoV-associated antigen. In contrast to the observed immunogenic role of ApoVs, the co-administration of unpulsed ApoVs with antigen-pulsed dendritic cells (DCs) significantly suppressed DC-mediated cytotoxic response in vivo; however, this occurred independent of CD169 expression. Overall, our results confirm that apoptosis contributes to both tolerance and immunity, as well as establishing CD169 as a critical mediator of the immune response to extracellular vesicles.

CD169 mediates the capture of exosomes in spleen and lymph node.

Exosomes are lipid nanovesicles released following fusion of the endosoma limiting membrane with the plasma membrane; however, their fate in lymphoid organs after their release remains controversial. We determined that sialoadhesin (CD169; Siglec-1) is required for the capture of B cell-derived exosomes via their surface-expressed α2,3-linked sialic acids. Exosome-capturing macrophages were present in the marginal zone of the spleen and in the subcapsular sinus of the lymph node. In vitro assays performed on spleen and lymph node sections confirmed that exosome binding to CD169 was not solely due to preferential fluid flow to these areas. Although the circulation half-life of exosomes in blood of wild-type and CD169(-/-) mice was similar, exosomes displayed altered distribution in CD169(-/-) mice, with exosomes freely accessing the outer marginal zone rim of SIGN-R1(+) macrophages and F4/80(+) red pulp macrophages. In the lymph node, exosomes were not retained in the subcapsular sinus of CD169(-/-) mice but penetrated deeper into the paracortex. Interestingly, CD169(-/-) mice demonstrated an enhanced response to antigen-pulsed exosomes. This is the first report of a role for CD169 in the capture of exosomes and its potential to mediate the immune response to exosomal antigen.

NK cells are required for dendritic cell-based immunotherapy at the time of tumor challenge.

Increasing evidence suggests that NK cells act to promote effective T cell-based antitumor responses. Using the B16-OVA melanoma model and an optimized Gram-positive bacteria-dendritic cell (DC) vaccination strategy, we determined that in vivo depletion of NK cells at time of tumor challenge abolished the benefit of DC immunotherapy. The contribution of NK cells to DC immunotherapy was dependent on tumor Ag presentation by DC, suggesting that NK cells act as helper cells to prime or reactivate tumor-specific T cells. The absence of NK cells at tumor challenge resulted in greater attenuation of tumor immunity than observed with selective depletion of either CD4 or CD8 T cell subsets. Although successful DC immunotherapy required IFN-γ, perforin expression was dispensable. Closer examination of the role of NK cells as helper cells in enhancing antitumor responses will reveal new strategies for clinical interventions using DC-based immunotherapy.

Altered transcription of murine genes induced in the small bowel by administration of probiotic strain Lactobacillus rhamnosus HN001.

Lactobacillus rhamnosus HN001 is a probiotic strain reported to increase resistance to epithelium-adherent and -invasive intestinal pathogens in experimental animals. To increase understanding of the relationship between strain HN001 and the bowel, transcription of selected genes in the mucosa of the murine small bowel was measured. Mice previously naive to lactobacilli (Lactobacillus-free mice) were examined after daily exposure to HN001 in drinking water. Comparisons were made to results from matched Lactobacillus-free mice. Infant and adult mice were investigated to provide a temporal view of gene expression in response to exposure to HN001. Genes sgk1, angptl4, and hspa1b, associated with the apoptosis pathway, were selected for investigation by reverse transcription-quantitative PCR on the basis of a preliminary duodenal DNA microarray screen. Normalized to gapdh gene transcription, these three genes were upregulated after 6 to 10 days exposure of adult mice to HN001. Angptl4 was shown by immunofluorescence to be upregulated in duodenal epithelial cells of mucosal samples. Epithelial cell migration was faster in HN001-exposed mice than in the Lactobacillus-free controls. Transcriptional responses in infant mice differed according to bowel region and age. For example, sgk1 was upregulated in duodenal, jejunal, and ileal mucosa of mice less than 25 days old, whereas angptl4 and hspa1b were upregulated at 10 days in the duodenum but downregulated in the jejunal mucosa until mice were 25 days old. Overall, the results provide links between a probiotic strain, mucosal gene expression, and host phenotype, which may be useful in delineating mechanisms of probiotic action.

Incorporation of triphenylphosphonium functionality improves the inhibitory properties of phenothiazine derivatives in Mycobacterium tuberculosis.

Tuberculosis (TB) is a difficult to treat disease caused by the bacterium Mycobacterium tuberculosis. The need for improved therapies is required to kill different M. tuberculosis populations present during infection and to kill drug resistant strains. Protein complexes associated with energy generation, required for the survival of all M. tuberculosis populations, have shown promise as targets for novel therapies (e.g., phenothiazines that target type II NADH dehydrogenase (NDH-2) in the electron transport chain). However, the low efficacy of these compounds and their off-target effects has made the development of phenothiazines as a therapeutic agent for TB limited. This study reports that a series of alkyltriphenylphosphonium (alkylTPP) cations, a known intracellular delivery functionality, improves the localization and effective concentration of phenothiazines at the mycobacterial membrane. AlkylTPP cations were shown to accumulate at biological membranes in a range of bacteria and lipophilicity was revealed as an important feature of the structure-function relationship. Incorporation of the alkylTPP cationic function significantly increased the concentration and potency of a series of phenothiazine derivatives at the mycobacterial membrane (the site of NDH-2), where the lead compound 3a showed inhibition of M. tuberculosis growth at 0.5µg/mL. Compound 3a was shown to act in a similar manner to that previously published for other active phenothiazines by targeting energetic processes (i.e., NADH oxidation and oxygen consumption), occurring in the mycobacterial membrane. This shows the enormous potential of alkylTPP cations to improve the delivery and therefore efficacy of bioactive agents targeting oxidative phosphorylation in the mycobacterial membrane.

Development of a compact bidirectional promoter-driven dual chimeric antigen receptor (CAR) construct targeting CD19 and CD20 in the Sleeping Beauty (SB) transposon system.

BACKGROUND: A bidirectional promoter-driven chimeric antigen receptor (CAR) cassette provides the simultaneous expression of two CARs, which significantly enhances dual antigen-targeted CAR T-cell therapy. METHODS: We developed a second-generation CAR directing CD19 and CD20 antigens, incorporating them in a head-to-head orientation from a bidirectional promoter using a single Sleeping Beauty transposon system. The efficacy of bidirectional promoter-driven dual CD19 and CD20 CAR T cells was determined in vitro against cell lines expressing either, or both, CD19 and CD20 antigens. In vivo antitumor activity was tested in Raji lymphoma-bearing immunodeficient NOD-scid IL2Rgammanull (NSG) mice. RESULTS: Of all tested promoters, the bidirectional EF-1α promoter optimally expressed transcripts from both sense (CD19-CAR) and antisense (GFP.CD20-CAR) directions. Superior cytotoxicity, cytokine production and antigen-specific activation were observed in vitro in the bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells. In contrast, a unidirectional construct driven by the EF-1α promoter, but using self-cleaving peptide-linked CD19 and CD20 CARs, showed inferior expression and in vitro function. Treatment of mice bearing advanced Raji lymphomas with bidirectional EF-1α promoter-driven CD19/CD20 CAR T cells effectively controlled tumor growth and extended the survival of mice compared with group treated with single antigen targeted CAR T cells. CONCLUSION: The use of bidirectional promoters in a single vector offers advantages of size and robust CAR expression with the potential to expand use in other forms of gene therapies like CAR T cells.

Optimal Annotation of Local Activation Time in Ventricular Tachycardia Substrate Mapping.

BACKGROUND: Accurate annotation of electrogram local activation time (LAT) is critical to the functional assessment of ventricular tachycardia (VT) substrate. Contemporary methods of annotation include: 1) earliest bipolar electrogram (LATearliest); 2) peak bipolar electrogram (LATpeak); 3) latest bipolar electrogram (LATlatest); and 4) steepest unipolar -dV/dt (LAT-dV/dt). However, no direct comparison of these methods has been performed in a large dataset, and it is unclear which provides the optimal functional analysis of the VT substrate. OBJECTIVES: This study sought to investigate the optimal method of LAT annotation during VT substrate mapping. METHODS: Patients with high-density VT substrate maps and a defined critical site for VT re-entry were included. All electrograms were annotated using 5 different methods: LATearliest, LATpeak, LATlatest, LAT-dV/dt, and the novel steepest unipolar -dV/dt using a dynamic window of interest (LATDWOI). Electrograms were also tagged as either late potentials and/or fractionated signals. Maps, utilizing each annotation method, were then compared in their ability to identify critical sites using deceleration zones. RESULTS: Fifty cases were identified with 1,.813 ± 811 points per map. Using LATlatest, a deceleration zone was present at the critical site in 100% of cases. There was no significant difference with LATearliest (100%) or LATpeak (100%). However, this number decreased to 54% using LAT-dV/dt and 76% for LATDWOI. Using LAT-dV/dt, only 33% of late potentials were correctly annotated, with the larger far field signals often annotated preferentially. CONCLUSIONS: Annotation with LAT-dV/dt and LATDWOI are suboptimal in VT substrate mapping. We propose that LATlatest should be the gold standard annotation method, as this allows identification of critical sites and is most suited to automation.

The impact of non-synonymous mutations on miRNA binding sites within the SARS-CoV-2 NSP3 and NSP4 genes.

Non-synonymous mutations in the SARS-CoV-2 spike region affect cell entry, tropism, and immune evasion, while frequent synonymous mutations may modify viral fitness. Host microRNAs, a type of non-coding RNA, play a crucial role in the viral life cycle, influencing viral replication and the host immune response directly or indirectly. Recently, we identified ten miRNAs with a high complementary capacity to target various regions of the SARS-CoV-2 genome. We filtered our candidate miRNAs to those only expressed with documented expression in SARS-CoV-2 target cells, with an additional focus on miRNAs that have been reported in other viral infections. We determined if mutations in the first SARS-CoV-2 variants of concern affected these miRNA binding sites. Out of ten miRNA binding sites, five were negatively impacted by mutations, with three recurrent synonymous mutations present in multiple SARS-CoV-2 lineages with high-frequency NSP3: C3037U and NSP4: G9802U/C9803U. These mutations were predicted to negatively affect the binding ability of miR-197-5p and miR-18b-5p, respectively. In these preliminary findings, using a dual-reporter assay system, we confirmed the ability of these miRNAs in binding to the predicted NSP3 and NSP4 regions and the loss/reduced miRNA bindings due to the recurrent mutations.

Retrospective Window of Interest Annotation Provides New Insights Into Functional Channels in Ventricular Tachycardia Substrate.

BACKGROUND: Accurate annotation of local activation time is crucial in the functional assessment of ventricular tachycardia (VT) substrate. A major limitation of modern mapping systems is the standard prospective window of interest (sWOI) is limited to 490 to 500 milliseconds, preventing annotation of very late potentials (LPs). A novel retrospective window of interest (rWOI), which allows annotation of all diastolic potentials, was used to assess the functional VT substrate. OBJECTIVES: This study sought to investigate the utility of a novel rWOI, which allows accurate visualization and annotation of all LPs during VT substrate mapping. METHODS: Patients with high-density VT substrate maps and a defined isthmus were included. All electrograms were manually annotated to latest activation using a novel rWOI. Reannotated substrate maps were correlated to critical sites, with areas of late activation examined. Propagation patterns were examined to assess the functional aspects of the VT substrate. RESULTS: Forty-eight cases were identified with 1,820 ± 826 points per map. Using the novel rWOI, 31 maps (65%) demonstrated LPs beyond the sWOI limit. Two distinct patterns of channel activation were seen during substrate mapping: 1) functional block with unidirectional conduction into the channel (76%); and 2) wave front collision within the channel (24%). In addition, a novel marker termed the zone of early and late crowding was studied in the rWOI substrate maps and found to have a higher positive predictive value (85%) than traditional deceleration zones (69%) for detecting critical sites of re-entry. CONCLUSIONS: The standard WOI of contemporary mapping systems is arbitrarily limited and results in important very late potentials being excluded from annotation. Future versions of electroanatomical mapping systems should provide longer WOIs for accurate local activation time annotation.

Directed Graph Mapping for Ventricular Tachycardia: A Comparison to Established Mapping Techniques.

BACKGROUND: Understanding underlying mechanism(s) and identifying critical circuit components are fundamental to successful ventricular tachycardia (VT) ablation. Directed graph mapping (DGM) offers a novel technique to identify the mechanism and critical components of a VT circuit. OBJECTIVES: This study sought to evaluate the accuracy of DGM in VT ablation compared with traditional mapping techniques and a commercially available automated conduction velocity mapping (ACVM) tool. METHODS: Patients with structural heart disease who had undergone a VT ablation with entrainment-proven critical isthmus and a high-density electroanatomical activation map were included. Traditional mapping (TM) consisted of a combination of local activation time and entrainment mapping and was considered the gold standard for determining the VT mechanism, circuit, and isthmus location. The same local activation time values were then processed using DGM and a commercially available ACVM (Coherent Mapping, Biosense Webster) tool. The aim of this study was to compare TM vs DGM and ACVM in their ability to identify the VT mechanism, characterize the VT circuit, and locate the critical isthmus. RESULTS: Thirty-five cases were identified. TM classified the VT mechanism as focal in 7 patients and re-entrant in 28 patients. TM classified 11 VTs as single-loop re-entry, 15 as dual-loop re-entry, 1 as complex, and 1 case was indeterminant. The overall agreement between DGM and TM for determining VT mechanism and circuit type was strong (kappa value = 0.79; P < 0.01), as was the agreement between ACVM and TM (kappa value = 0.66; P < 0.01). Both DGM and ACVM identified the putative VT isthmus in 25 (89%) of the re-entrant cases. Focal activation was correctly identified by both techniques in all cases. CONCLUSIONS: DGM is a rapid automated algorithm that has a strong level of agreement with TM for manually re-annotated VT maps.

Multiple traces of monkeypox detected in non-sewered wastewater with sparse sampling from a densely populated metropolitan area in Asia.

The monkeypox virus is excreted in the feces of infected individuals. Therefore, there is an interest in using viral load detection in wastewater for sentinel early surveillance at a community level and as a complementary approach to syndromic surveillance. We collected wastewater from 63 sewered and non-sewered locations in Bangkok city center between May and August 2022. Monkeypox viral DNA copy numbers were quantified using real-time polymerase chain reaction (PCR) and confirmed positive by Sanger sequencing. Monkeypox viral DNA was first detected in wastewater from the second week of June 2022, with a mean copy number of 16.4 copies/ml (n = 3). From the first week of July, the number of viral DNA copies increased to a mean copy number of 45.92 copies/ml. Positive samples were Sanger sequenced and confirmed the presence of the monkeypox virus. Our study is the first to detect monkeypox viral DNA in wastewater from various locations within Thailand. Results suggest that this could be a complementary source for detecting viral DNA and predicting upcoming outbreaks.