Small Molecules Co-targeting CKIα and the Transcriptional Kinases CDK7/9 Control AML in Preclinical Models.

CKIα ablation induces p53 activation, and CKIα degradation underlies the therapeutic effect of lenalidomide in a pre-leukemia syndrome. Here we describe the development of CKIα inhibitors, which co-target the transcriptional kinases CDK7 and CDK9, thereby augmenting CKIα-induced p53 activation and its anti-leukemic activity. Oncogene-driving super-enhancers (SEs) are highly sensitive to CDK7/9 inhibition. We identified multiple newly gained SEs in primary mouse acute myeloid leukemia (AML) cells and demonstrate that the inhibitors abolish many SEs and preferentially suppress the transcription elongation of SE-driven oncogenes. We show that blocking CKIα together with CDK7 and/or CDK9 synergistically stabilize p53, deprive leukemia cells of survival and proliferation-maintaining SE-driven oncogenes, and induce apoptosis. Leukemia progenitors are selectively eliminated by the inhibitors, explaining their therapeutic efficacy with preserved hematopoiesis and leukemia cure potential; they eradicate leukemia in MLL-AF9 and Tet2-/-;Flt3ITD AML mouse models and in several patient-derived AML xenograft models, supporting their potential efficacy in curing human leukemia.

Immunity in acute myeloid leukemia: Where the immune response and targeted therapy meet.

Acute myeloid leukemia (AML) is a highly aggressive disease with high relapse and mortality rates. Recent years have shown a surge in novel therapeutic development for AML, both in clinical and preclinical stages. These developments include targeted therapies based on AML-specific molecular signatures as well as more general immune modulation and vaccination studies. In this review, we will explore the evolving arena of AML therapy and suggest some intriguing connections between immune system modulation and targeted therapy. Improved understanding of the immune system involvement in various stages of the disease and the crosstalk between immune effectors, targeted therapy, and AML cells can provide a better framework for designing the next generation of AML therapies.

More extreme duplication in FDA Adverse Event Reporting System detected by literature reference normalization and fuzzy string matching.

PURPOSE: Literature reports of adverse drug events can be replicated across multiple companies, resulting in extreme duplication (defined as a majority of reports being duplicates) in the FDA Adverse Event Reporting System (FAERS) database because they can escape legacy duplicate detection algorithms routinely deployed on that data source. Literature reference field, added to in 2014, could potentially be utilized to identify replicated reports. FAERS does not enforce adherence to the Vancouver referencing convention, thus the same article may be referenced differently leading to duplication. The objective of this analysis is to determine if variations of the same literature references observed in FAERS can be resolved with text normalization and fuzzy string matching. METHODS: We normalized the literature references recorded in the FAERS database through the first quarter of 2021 with a rule-based algorithm so that they better conform to the Vancouver convention. Levenshtein distance was then utilized to merge sufficiently similar normalized literature references together. RESULTS: Normalization of literature references increases the percentage that can be parsed into author, title, and journal from 61.74% to 93.93%. We observe that about 98% of pairs within groups do have a Levenshtein similarity of the title above the threshold. The extreme duplication ranged from 66% to 87% with a median of 72% of reports being duplicates and often involved addictovigilance scenarios. CONCLUSIONS: We have shown that these normalized references can be merged via fuzzy string matching to improve enumeration of all the individual case safety reports that refer to the same article. Inclusion of the PubMed ID and adherence to the Vancouver convention could facilitate identification of duplicates in the FAERS dataset. Awareness of this phenomenon may improve disproportionality analysis, especially in areas such as addictovigilance.

First cases of SARS-CoV-2 BA.2.86 in Denmark, 2023.

We describe 10 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant BA.2.86 detected in Denmark, including molecular characteristics and results from wastewater surveillance that indicate that the variant is circulating in the country at a low level. This new variant with many spike gene mutations was classified as a variant under monitoring by the World Health Organization on 17 August 2023. Further global monitoring of COVID-19, BA.2.86 and other SARS-CoV-2 variants is highly warranted.

Maternal paracetamol intake and fetal ductus arteriosus constriction/closure: comprehensive signal evaluation using the Austin Bradford Hill criteria.

PURPOSE: Acetaminophen (APAP) is available over-the-counter and widely regarded as safe for use in pregnancy. APAP has been used to close a persistently patent ductus arteriosus. Fetal constriction/closure of the ductus arteriosus (FCCDA), of public health interest given the drug's widespread use during pregnancy, is being monitored globally, including by the European Medicines Agency Pharmacovigilance Risk Assessment Committee. Our objective was to share a comprehensive signal evaluation of FCCDA with in utero APAP exposure to determine if the totality of evidence is sufficiently more consistent with one of the following two possibilities: (1) APAP never contributes to FCCDA (null hypothesis or HO) versus (2) APAP may in some cases be at least a contributory cause of in utero DA narrowing (alternative hypothesis or HA) to justify risk communication. METHODS: To assess the relative support for HO versus HA, we synthesize and interpret within an Austin Bradford Hill criteria framework a comprehensive, cross-disciplinary set of published information and de novo analysis, including toxicology, epidemiology, clinical pharmacology, and clinical and quantitative pharmacovigilance analysis of spontaneous reports. RESULTS: While residual uncertainty remains, the totality of information is more compatible with HA than H0, to the extent that it is reasonably possible that APAP may sometimes be at least a contributory cause of FCCDA. CONCLUSION: It is reasonably possible that APAP may sometimes be at least a contributory cause of FCCDA, and this should therefore be communicated to stakeholders. TRIAL REGISTRATION: CLINICALTRIALS. GOV REGISTRATION: NOT APPLICABLE.

Incidence of venous and arterial thromboembolic events reported in the tofacitinib rheumatoid arthritis, psoriasis and psoriatic arthritis development programmes and from real-world data.

OBJECTIVES: Tofacitinib is a Janus kinase inhibitor for the treatment of rheumatoid arthritis (RA), psoriatic arthritis (PsA) and ulcerative colitis, and has been investigated in psoriasis (PsO). Routine pharmacovigilance of an ongoing, open-label, blinded-endpoint, tofacitinib RA trial (Study A3921133; NCT02092467) in patients aged ≥50 years and with ≥1 cardiovascular risk factor identified a higher frequency of pulmonary embolism (PE) and all-cause mortality for patients receiving tofacitinib 10 mg twice daily versus those receiving tumour necrosis factor inhibitors and resulted in identification of a safety signal for tofacitinib. Here, we report the incidence of deep vein thrombosis (DVT), PE, venous thromboembolism (VTE; DVT or PE) and arterial thromboembolism (ATE) from the tofacitinib RA (excluding Study A3921133), PsA and PsO development programmes and observational studies. Data from an ad hoc safety analysis of Study A3921133 are reported separately within. METHODS: This post-hoc analysis used data from separate tofacitinib RA, PsO and PsA programmes. Incidence rates (IRs; patients with events per 100 patient-years' exposure) were calculated for DVT, PE, VTE and ATE, including for populations stratified by defined baseline cardiovascular or VTE risk factors. Observational data from the US Corrona registries (including cardiovascular risk factor stratification), IBM MarketScan research database and the US FDA Adverse Event Reporting System (FAERS) database were analysed. RESULTS: 12 410 tofacitinib-treated patients from the development programmes (RA: n=7964; PsO: n=3663; PsA: n=783) were included. IRs (95% CI) of thromboembolic events among the all tofacitinib cohorts' average tofacitinib 5 mg and 10 mg twice daily treated patients for RA, respectively, were: DVT (0.17 (0.09-0.27) and 0.15 (0.09-0.22)); PE (0.12 (0.06-0.22) and 0.13 (0.08-0.21)); ATE (0.32 (0.22-0.46) and 0.38 (0.28-0.49)). Among PsO patients, IRs were: DVT (0.06 (0.00-0.36) and 0.06 (0.02-0.15)); PE (0.13 (0.02-0.47) and 0.09 (0.04-0.19)); ATE (0.52 (0.22-1.02) and 0.22 (0.13-0.35)). Among PsA patients, IRs were: DVT (0.00 (0.00-0.28) and 0.13 (0.00-0.70)); PE (0.08 (0.00-0.43) and 0.00 (0.00-0.46)); ATE (0.31 (0.08-0.79) and 0.38 (0.08-1.11)). IRs were similar between tofacitinib doses and generally higher in patients with baseline cardiovascular or VTE risk factors. IRs from the overall Corrona populations and in Corrona RA patients (including tofacitinib-naïve/biologic disease-modifying antirheumatic drug-treated and tofacitinib-treated) with baseline cardiovascular risk factors were similar to IRs observed among the corresponding patients in the tofacitinib development programme. No signals of disproportionate reporting of DVT, PE or ATE with tofacitinib were identified in the FAERS database. CONCLUSIONS: DVT, PE and ATE IRs in the tofacitinib RA, PsO and PsA programmes were similar across tofacitinib doses, and generally consistent with observational data and published IRs of other treatments. As expected, IRs of thromboembolic events were elevated in patients with versus without baseline cardiovascular or VTE risk factors, and were broadly consistent with those observed in the Study A3921133 ad hoc safety analysis data, although the IR (95% CI) for PE was greater in patients treated with tofacitinib 10 mg twice daily in Study A3921133 (0.54 (0.32-0.87)), versus patients with baseline cardiovascular risk factors treated with tofacitinib 10 mg twice daily in the RA programme (0.24 (0.13-0.41)).

Contralateral breast cancer can represent a metastatic spread of the first primary tumor: determination of clonal relationship between contralateral breast cancers using next-generation whole genome sequencing.

INTRODUCTION: By convention, a contralateral breast cancer (CBC) is treated as a new primary tumor, independent of the first cancer (BC1). Although there have been indications that the second tumor (BC2) sometimes may represent a metastatic spread of BC1, this has never been conclusively shown. We sought to apply next-generation sequencing to determine a "genetic barcode" for each tumor and reveal the clonal relationship of CBCs. METHODS: Ten CBC patients with detailed clinical information and available fresh frozen tumor tissue were studied. Using low-coverage whole genome DNA-sequencing data for each tumor, chromosomal rearrangements were enumerated and copy number profiles were generated. Comparisons between tumors provided an estimate of clonal relatedness for tumor pairs within individual patients. RESULTS: Between 15-256 rearrangements were detected in each tumor (median 87). For one patient, 76 % (68 out of 90) of the rearrangements were shared between BC1 and BC2, highly consistent with what has been seen for true primary-metastasis pairs (>50 %) and thus confirming a common clonal origin of the two tumors. For most of the remaining cases, BC1 and BC2 had similarly low overlap as unmatched randomized pairs of tumors from different individuals, suggesting the CBC to represent a new independent primary tumor. CONCLUSION: Using rearrangement fingerprinting, we show for the first time with certainty that a contralateral BC2 can represent a metastatic spread of BC1. Given the poor prognosis of a generalized disease compared to a new primary tumor, these women need to be identified at diagnosis of CBC for appropriate determination of treatment. Our approach generates a promising new method to assess clonal relationship between tumors. Additional studies are required to confirm the frequency of CBCs representing metastatic events.

Identification of up- and down-regulated proteins in doxorubicin-resistant uterine cancer cells: reticulocalbin-1 plays a key role in the development of doxorubicin-associated resistance.

Drug resistance is a frequent cause of failure in cancer chemotherapy treatments. In this study, a pair of uterine sarcoma cancer lines, MES-SA, and doxorubicin-resistant partners, MES-SA/DxR-2µM cells and MES-SA/DxR-8µM cells, as a model system to investigate resistance-dependent proteome alterations and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to perform this research and the results revealed that doxorubicin-resistance altered the expression of 208 proteins in which 129 identified proteins showed dose-dependent manners in response to the levels of resistance. Further studies have used RNA interference, H2A.X phosphorylation assay, cell viability analysis, and analysis of apoptosis against reticulocalbin-1 (RCN1) proteins, to prove its potency on the formation of doxorubicin resistance as well as the attenuation of doxorubicin-associated DNA double strand breakage. To sum up, our results provide useful diagnostic markers and therapeutic candidates such as RCN1 for the treatment of doxorubicin-resistant uterine cancer.

Assessing the extent and impact of the masking effect of disproportionality analyses on two spontaneous reporting systems databases.

BACKGROUND: Masking is a statistical issue by which signals are hidden by the presence of other medicines in the database. In the absence algorithm, the impact of the masking effect has not been fully investigated. OBJECTIVE: Our study is aimed at assessing the extent and the impact of the masking effect on two large spontaneous reporting databases. STUDY DESIGN: Cross sectional study using a set of terms of importance for public health in two spontaneous reporting databases. SETTING: The analyses were performed on EudraVigilance (EV) and the Pfizer spontaneous reporting database (PfDB). MAIN OUTCOME MEASURE: Using the masking ratio, we have identified and removed the products inducing the highest masking effect. RESULTS: Studying a total of almost 50 000 drug-event combinations masking had an impact on approximately 60% of drug-event combinations were masked by another product with a masking ratio >1 in EV and 84% in PfDB. The prevalence of important masking was quite rare (0.003% of the DECs) and mainly affected events rarely reported in EV. The products involved in the highest masking effects are products known to induce the reaction. The removal of the masking effect of the highest masking product has revealed 974 signals of disproportionate reporting in EV including true signals. The study shows that the original ranking provided by the quantitative methods included in our study is marginally affected by the removal of the masking product. CONCLUSION: Our study suggests that significant masking is rare in large spontaneous databases and mostly affects events rarely reported in EV.

A conceptual approach to the masking effect of measures of disproportionality.

BACKGROUND: Masking is a statistical issue by which true signals of disproportionate reporting are hidden by the presence of other products in the database. Masking is currently not perfectly understood. There is no algorithm to identify the potential masking drugs to remove them for subsequent analyses of disproportionality. OBJECTIVE: The primary objective of our study is to develop a mathematical framework for assessing the extent and impact of the masking effect of measures of disproportionality. METHOD: We have developed a masking ratio that quantifies the masking effect of a given product. We have conducted a simulation study to validate our algorithm. RESULTS: The masking ratio is a measure of the strength of the masking effect whether the analysis is performed at the report or event level, and the manner in which reports are allocated to cells in the contingency table significantly impact the masking mechanisms. The reports containing both the product of interest and the masking product need to be handled appropriately. The proposed algorithm can use simplified masking provided that underlying assumptions (in particular the size of the database) are verified. For any event, the strongest masking effect is associated with the drug with the highest number of records (reports excluding the product of interest). CONCLUSION: Our study provides significant insights with practical implications for real-world pharmacovigilance that are supported by both real and simulated data. The public health impact of masking is still unknown.

Potential Signals of COVID-19 as an Effect Modifier of Adverse Drug Reactions.

PURPOSE: COVID-19 infection may interact with patients' medical conditions or medications. The objective of this study was to identify potential signals of effect modification of adverse drug reactions by statistical reporting interactions with COVID-19 infection (SRIsCOVID-19) in a large spontaneous reporting database. METHODS: Data from the US Food and Drug Administration Adverse Event Reporting System through the second quarter of 2020 were used. Three-dimensional disproportionality analyses were conducted to identify drug-event-event (DEE) combinations, for which 1 of the events was COVID-19 infection, that were disproportionately reported. Effect size was quantified by an interaction signal score (INTSS) when COVID-19 was coreported as an adverse event or an indication (INTSSCOVID-19). An SRICOVID-19 exists when the calculated INTSSCOVID-19 is >2. The analyses focused on pandemic-emergent SRIsCOVID-19. Screening for extreme duplication of cases was applied. To assess possible reporting artifacts during the early pandemic as an alternative explanation for pandemic-emergent SRICOVID-19, we repeated the analyses with an additional year of data to gauge temporal stability of our findings. FINDINGS: When examining DEE interactions, 193 emergent SRIsCOVID-19 were identified, involving 44 drugs and 88 events, in addition to COVID-19 infection. Of the 44 drugs recorded, most were immunosuppressant or modulatory drugs, followed by antivirals. Seven drugs (eg, azithromycin) were identified in emergent SRIsCOVID-19 with preferred terms representing off-label use for prevention or treatment of COVID-19 infection. These drugs were in fact repurposed for COVID-19 treatment, supporting assay sensitivity of our procedure. Infections and infestations were the most frequently observed system organ class, followed by the general disorders and respiratory disorders. The psychiatric system organ class had only a few emergent SRIsCOVID-19 but contained the largest INTSSs. Less commonly reported manifestations of COVID-19 (e.g., skin events) were also identified. After excluding DEE combinations that were highly suggestive of extreme duplication, there remained a more robust set of emergent SRIsCOVID-19, which were supported by biological plausibility considerations. Our findings indicate a relative temporal stability, with >90% of SRIsCOVID-19 persisting after updating the analysis with an additional year of data. IMPLICATIONS: The signals identified in the analyses could be critical in refining our understanding of the causality of spontaneously reported adverse drug events and thus informing the ongoing care of patients with COVID-19. Our findings also underscore the importance of undetected report duplication as a distorting influence on disproportionality analysis.

Effects of self-efficacy on frontal midline theta power and golf putting performance.

Introduction: Self-efficacy (SE), defined as an individual's belief in their ability to complete a task, is linked to top-down attentional control, influencing motor performance in sports. Although the behavioral effects of SE are well-documented, there is a lack of research on the mechanisms through which SE affects sports performance. Our research aims to elucidate the neurophysiological mechanisms that underlie the impact of self-efficacy on sports performance. Specifically, we intend to explore the effects of low and high SE on frontal midline theta (Fmθ) activity, associated with sustained top-down attention, and on motor performance. Methods: We recruited thirty-four professional golfers to perform 60 putts, during which their electroencephalographic activity was monitored. SE levels were assessed using a visual analog scale from 0 to 10 before each putt, with scores categorized into higher or lower SE based on each golfer's individual average score. Results: Paired t-tests indicated that trials with higher SE scores had a higher putting success rate than those with lower SE scores (53.3% vs. 46.7%). Furthermore, trials associated with higher SE scores exhibited lower Fmθ activity compared to those with lower SE scores (4.49 vs. 5.18). Discussion: Our results suggest that higher SE is associated with reduced top-down attentional control, leading to improved putting performance. These findings support Bandura's theory of SE, which suggests that the effects of efficacy beliefs are mediated by cognitive, motivational, emotional, and decision-making processes. This study sheds light on the intermediate processes of SE by examining its impact on the anticipation of outcomes, sports performance, and attentional control prior to putting.

Variations in the persistence of 5'-end genomic and subgenomic SARS-CoV-2 RNAs in wastewater from aircraft, airports and wastewater treatment plants.

Wastewater sequencing has become a powerful supplement to clinical testing in monitoring SARS-CoV-2 infections in the post-COVID-19 pandemic era. While its applications in measuring the viral burden and main circulating lineages in the community have proved their efficacy, the variations in sequencing quality and coverage across the different regions of the SARS-CoV-2 genome are not well understood. Furthermore, it is unclear how different sample origins, viral extraction and concentration methods and environmental factors impact the reads sequenced from wastewater. Using high-coverage, amplicon-based, paired-end read sequencing of viral RNA extracted from wastewater collected directly from aircraft, pooled from different aircraft and airport buildings or from regular wastewater plants, we assessed the genome coverage across the sample groups with a focus on the 5'-end region covering the leader sequence and investigated whether it was possible to detect subgenomic RNA from viral material recovered from wastewater. We identified distinct patterns in the persistence of the different genomic regions across the different types of wastewaters and the existence of chimeric reads mapping to non-amplified regions. Our findings suggest that preservation of the 5'-end of the genome and the ability to detect subgenomic RNA reads, though highly susceptible to environment and sample processing conditions, may be indicative of the quality and amount of the viral RNA present in wastewater.

Pneumothorax as an adverse drug event: an exploratory aggregate analysis of the US FDA AERS database including a confounding by indication analysis inspired by Cornfield's condition.

INTRODUCTION: Pneumothorax is either primary or secondary. Secondary pneumothorax is usually due to trauma, including various non-pharmacologic iatrogenic triggers. Although not normally thought of as an adverse drug event (ADE) secondary pneumothorax is associated with numerous drugs, though it is often difficult to determine whether this association is causal in nature, or reflects an epiphenomenon of efficacy or inefficacy, or confounding by indication (CBI). Herein we explore this association in a large health authority drug safety surveillance database. METHODS: A quantitative pharmacovigilance (PhV) methodology known as disproportionality analysis was applied to the United States Food and Drug Administration (US FDA) Adverse Event Reporting System (AERS) database to explore the quantitative reporting dependencies between drugs and the adverse event pneumothorax as well the corresponding reporting dependencies between drugs and reported indications that may be risk factors for pneumothorax themselves in order to explore the potential contribution of CBI. RESULTS: We found 1. Multiple drugs are associated with pneumothorax; 2. Surfactants and oncology drugs account for most statistically distinctive associations with pneumothorax; 3. Pulmonary surfactants, pentamidine and nitric oxide have the largest statistical reporting associations 4. CBI may play a prominent role in reports of drug-associated pneumothorax. CONCLUSIONS: Disproportionality analysis (DA) can provide insights into the spontaneous reporting dependencies between drugs and pneumothorax. CBI assessment based on DA and Cornfield's inequality presents an additional novel option for the initial exploration of potential safety signals in PhV.

Sonographic examination of the buttock.

The buttock is a common site of pathology and ultrasound and is often the first-line imaging modality to examine soft tissue lesions of the buttock region. This review describes the ultrasound technique used, the relevant ultrasound anatomy, and the sonographic appearances of common and uncommon pathological conditions found in the buttock region.

Anti-tumor Effects of IL-1ß Induced TRAIL-Expressing hUCMSCs on Embelin Treated Breast Cancer Cell Lines.

BACKGROUND: Human umbilical cord mesenchymal stem cells (hUCMSCs) have high therapeutic value in cancer treatment. We have found that pre-activating hUCMSCs with IL-1ß promotes tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression and facilitates anti-tumor effect. Furthermore, embelin has been found to induce apoptosis of different cancer cell lines by upregulating the expression of TRAIL receptor 1 (DR4) and TRAIL receptor 2 (DR5). This study investigated whether IL-1ß induced TRAIL-expressing hUCMSCs, in combination with low-dose embelin, could further induce apoptosis in breast cancer cell lines. MATERIALS AND METHODS: MTT assay was used to examine the cytotoxicity of embelin in MDA-MB-231 and MCF-7. To detect the interested protein expression in cells, Western blot and cell immunofluorescence were used to double-confirm the observed results. Annexin V/PI apoptosis assay was detected by flow cytometry to analyze the apoptosis rate of embelin treated breast cancer cell lines and the effect of co-culturing with breast cancer cells and hUCMSCs. RESULTS: Using Western blot and immunofluorescence, we found that breast cancer cell lines treated with low-dose embelin (2.5-5 µM) increased the expression of apoptosis-related receptor DR4, DR5 and the cleaved caspase 8, 9 and 3. Moreover, TRAIL expression was enhanced in IL-1ß induced hUCMSCs. Combining these observations, we expected that coculturing IL-1ß induced hUCMSCs with low dose embelin treated MDA-MB-231 and MCF-7 cells might enhance the apoptosis of breast cancer cells. We confirmed via flow cytometry that coculture of IL-1ß induced TRAIL-expressing hUCMSCs and embelin treated MDA-MB-231 and MCF-7 cells enhances the apoptosis rate of these breast cancer cells. CONCLUSION: We found that embelin upregulated the expression of DR4 and DR5 to increase the TRAIL-mediated apoptosis in breast cancer cell lines. Low dose embelin treated breast cancer cell lines in combination with IL-1ß induced TRAIL-expressing hUCMSCs may become a potential anti-tumor therapy.

Comparative subgenomic mRNA profiles of SARS-CoV-2 Alpha, Delta and Omicron BA.1, BA.2 and BA.5 sub-lineages using Danish COVID-19 genomic surveillance data.

BACKGROUND: Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly spread worldwide in the population since it was first detected in late 2019. The transcription and replication of coronaviruses, although not fully understood, is characterised by the production of genomic length RNA and shorter subgenomic RNAs to make viral proteins and ultimately progeny virions. Observed levels of subgenomic RNAs differ between sub-lineages and open reading frames but their biological significance is presently unclear. METHODS: Using a large and diverse panel of virus sequencing data produced as part of the Danish COVID-19 routine surveillance together with information in electronic health registries, we assessed the association of subgenomic RNA levels with demographic and clinical variables of the infected individuals. FINDINGS: Our findings suggest no significant statistical relationship between levels of subgenomic RNAs and host-related factors. INTERPRETATION: Differences between lineages and subgenomic ORFs may be related to differences in target cell tropism, early virus replication/transcription kinetics or sequence features. FUNDING: The author(s) received no specific funding for this work.

Unveiling Rare Pathogens and Antibiotic Resistance in Tanzanian Cholera Outbreak Waters.

The emergence of antibiotic resistance is a global health concern. Therefore, understanding the mechanisms of its spread is crucial for implementing evidence-based strategies to tackle resistance in the context of the One Health approach. In developing countries where sanitation systems and access to clean and safe water are still major challenges, contamination may introduce bacteria and bacteriophages harboring antibiotic resistance genes (ARGs) into the environment. This contamination can increase the risk of exposure and community transmission of ARGs and infectious pathogens. However, there is a paucity of information on the mechanisms of bacteriophage-mediated spread of ARGs and patterns through the environment. Here, we deploy Droplet Digital PCR (ddPCR) and metagenomics approaches to analyze the abundance of ARGs and bacterial pathogens disseminated through clean and wastewater systems. We detected a relatively less-studied and rare human zoonotic pathogen, Vibrio metschnikovii, known to spread through fecal--oral contamination, similarly to V. cholerae. Several antibiotic resistance genes were identified in both bacterial and bacteriophage fractions from water sources. Using metagenomics, we detected several resistance genes related to tetracyclines and beta-lactams in all the samples. Environmental samples from outlet wastewater had a high diversity of ARGs and contained high levels of blaOXA-48. Other identified resistance profiles included tetA, tetM, and blaCTX-M9. Specifically, we demonstrated that blaCTX-M1 is enriched in the bacteriophage fraction from wastewater. In general, however, the bacterial community has a significantly higher abundance of resistance genes compared to the bacteriophage population. In conclusion, the study highlights the need to implement environmental monitoring of clean and wastewater to inform the risk of infectious disease outbreaks and the spread of antibiotic resistance in the context of One Health.