Linezolid-resistant Enterococcus faecium clinical isolates from Pakistan: a genomic analysis.

BACKGROUND: Linezolid-resistant Enterococcus faecium (LRE) is a global priority pathogen. Thirteen LRE were reported from clinical specimens between November 2021 and April 2023 at two laboratories in Karachi, Pakistan. We aimed to investigate the strain types and genes associated with linezolid resistance among these isolates. Whole genome sequencing (WGS) was performed and analyzed by multilocus sequence typing (MLST). The presence of linezolid resistance genes was identified using ResFinder v4.1.11 and the LRE-finder tool. RESULTS: Twelve isolates belonged to clonal complex 17 (CC17); ST80 (n = 10), ST612 (n = 1) and ST1380 (n = 1). Six isolates showed the presence of optrA gene and G2576T mutations in the 23S rRNA gene, while six showed poxtA and cfr(D) genes. One isolate showed the combination of optrA, cfr(D) and poxtA genes. CONCLUSION: Our findings show the circulation of CC17 sequence types with a known outbreak potential and we identified molecular mechanisms of resistance that were not previously reported from Pakistan.

The global population stru cture of Lacticaseibacillus rhamnosus and its application to an investigation of a rare case of infective endocarditis.

BACKGROUND: Lacticaseibacillus (formerly Lactobacillus) rhamnosus is widely used in probiotics or food supplements to promote microbiome health and may also be part of the normal microbiota of the human gastrointestinal tract. However, it rarely also causes invasive or severe infections in patients. It has been postulated that these infections may originate from probiotics or from endogenous commensal reservoirs. In this report, we examine the population structure of Lacticaseibacillus rhamnosus and investigate the utility of using bacterial genomics to identify the source of invasive Lacticaseibacillus infections. METHODS: Core genome phylogenetic analysis was performed on 602 L. rhamnosus genome sequences from the National Center for Biotechnology public database. This information was then used along with newly generated sequences of L. rhamnosus isolates from yogurt to investigate a fatal case of L. rhamnosus endocarditis. RESULTS: Phylogenetic analysis demonstrated substantial genetic overlap of L. rhamnosus isolates cultured from food, probiotics, infected patients, and colonized individuals. This was applied to a patient who had both consumed yogurt and developed L. rhamnosus endocarditis to attempt to identify the source of his infection. The sequence of the isolate from the patient's bloodstream differed at only one nucleotide position from one of the yogurt isolates. Both isolates belonged to a clade, identified here as clade YC, composed of mostly gastrointestinal isolates from healthy individuals, some of which also differed by only a single nucleotide change from the patient's isolate. CONCLUSIONS: As illustrated by this case, whole genome sequencing may be insufficient to reliably determine the source of invasive infections caused by L. rhamnosus.

Evolution of SARS-CoV-2 in the murine central nervous system drives viral diversification.

Severe coronavirus disease 2019 and post-acute sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are associated with neurological complications that may be linked to direct infection of the central nervous system (CNS), but the selective pressures ruling neuroinvasion are poorly defined. Here we assessed SARS-CoV-2 evolution in the lung versus CNS of infected mice. Higher levels of viral divergence were observed in the CNS than the lung after intranasal challenge with a high frequency of mutations in the spike furin cleavage site (FCS). Deletion of the FCS significantly attenuated virulence after intranasal challenge, with lower viral titres and decreased morbidity compared with the wild-type virus. Intracranial inoculation of the FCS-deleted virus, however, was sufficient to restore virulence. After intracranial inoculation, both viruses established infection in the lung, but dissemination from the CNS to the lung required the intact FCS. Cumulatively, these data suggest a critical role for the FCS in determining SARS-CoV-2 tropism and compartmentalization.

Distinctive evolution of alveolar T cell responses is associated with clinical outcomes in unvaccinated patients with SARS-CoV-2 pneumonia.

The evolution of T cell molecular signatures in the distal lung of patients with severe pneumonia is understudied. Here, we analyzed T cell subsets in longitudinal bronchoalveolar lavage fluid samples from 273 patients with severe pneumonia, including unvaccinated patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or with respiratory failure not linked to pneumonia. In patients with SARS-CoV-2 pneumonia, activation of interferon signaling pathways, low activation of the NF-κB pathway and preferential targeting of spike and nucleocapsid proteins early after intubation were associated with favorable outcomes, whereas loss of interferon signaling, activation of NF-κB-driven programs and specificity for the ORF1ab complex late in disease were associated with mortality. These results suggest that in patients with severe SARS-CoV-2 pneumonia, alveolar T cell interferon responses targeting structural SARS-CoV-2 proteins characterize individuals who recover, whereas responses against nonstructural proteins and activation of NF-κB are associated with poor outcomes.

Surveillance Metrics and History of the COVID-19 Pandemic in Central Asia: Updated Epidemiological Assessment.

BACKGROUND: This study updates the COVID-19 pandemic surveillance in Central Asia we conducted during the first year of the pandemic by providing 2 additional years of data for the region. The historical context provided through additional data can inform regional preparedness and early responses to infectious outbreaks of either the SARS-CoV-2 virus or future pathogens in Central Asia. OBJECTIVE: First, we aim to measure whether there was an expansion or contraction in the pandemic in Central Asia when the World Health Organization (WHO) declared the end of the public health emergency for the COVID-19 pandemic on May 5, 2023. Second, we use dynamic and genomic surveillance methods to describe the history of the pandemic in the region and situate the window of the WHO declaration within the broader history. Third, we aim to provide historical context for the course of the pandemic in Central Asia. METHODS: Traditional surveillance metrics, including counts and rates of COVID-19 transmissions and deaths, and enhanced surveillance indicators, including speed, acceleration, jerk, and persistence, were used to measure shifts in the pandemic. To identify the appearance and duration of variants of concern, we used data on sequenced SARS-CoV-2 variants from the Global Initiative on Sharing All Influenza Data (GISAID). We used Nextclade nomenclature to collect clade designations from sequences and Pangolin nomenclature for lineage designations of SARS-CoV-2. Finally, we conducted a 1-sided t test to determine whether regional speed was greater than an outbreak threshold of 10. We ran the test iteratively with 6 months of data across the sample period. RESULTS: Speed for the region had remained below the outbreak threshold for 7 months by the time of the WHO declaration. Acceleration and jerk were also low and stable. Although the 1- and 7-day persistence coefficients remained statistically significant, the coefficients were relatively small in magnitude (0.125 and 0.347, respectively). Furthermore, the shift parameters for either of the 2 most recent weeks around May 5, 2023, were both significant and negative, meaning the clustering effect of new COVID-19 cases became even smaller in the 2 weeks around the WHO declaration. From December 2021 onward, Omicron was the predominant variant of concern in sequenced viral samples. The rolling t test of speed equal to 10 became entirely insignificant for the first time in March 2023. CONCLUSIONS: Although COVID-19 continues to circulate in Central Asia, the rate of transmission remained well below the threshold of an outbreak for 7 months ahead of the WHO declaration. COVID-19 appeared to be endemic in the region and no longer reached the threshold of a pandemic. Both standard and enhanced surveillance metrics suggest the pandemic had ended by the time of the WHO declaration.

South Asia's COVID-19 History and Surveillance: Updated Epidemiological Assessment.

BACKGROUND: This study updates our findings from the COVID-19 pandemic surveillance we first conducted in South Asia in 2020 with 2 additional years of data for the region. We assess whether COVID-19 had transitioned from pandemic to endemic at the point the World Health Organization (WHO) ended the public health emergency status for COVID-19 on May 5, 2023. OBJECTIVE: First, we aim to measure whether there was an expansion or contraction in the pandemic in South Asia around the WHO declaration. Second, we use dynamic and genomic surveillance methods to describe the history of the pandemic in the region and situate the window of the WHO declaration within the broader history. Third, we aim to provide historical context for the course of the pandemic in South Asia. METHODS: In addition to updating the traditional surveillance data and dynamic panel estimates from our original study, this study used data on sequenced SARS-CoV-2 variants from the Global Initiative on Sharing All Influenza Data (GISAID) to identify the appearance and duration of variants of concern. We used Nextclade nomenclature to collect clade designations from sequences and Pangolin nomenclature for lineage designations of SARS-CoV-2. Finally, we conducted a 1-sided t test to determine whether regional weekly speed or transmission rate per 100,000 population was greater than an outbreak threshold of 10. We ran the test iteratively with 6 months of data across the sample period. RESULTS: Speed for the region had remained below the outbreak threshold for over a year by the time of the WHO declaration. Acceleration and jerk were also low and stable. While the 1-day persistence coefficients remained statistically significant and positive (1.168), the 7-day persistence coefficient was negative (-0.185), suggesting limited cluster effects in which cases on a given day predict cases 7 days forward. Furthermore, the shift parameters for either of the 2 most recent weeks around May 5, 2023, did not indicate any overall change in the persistence measure around the time of the WHO declaration. From December of 2021 onward, Omicron was the predominant variant of concern in sequenced viral samples. The rolling t test of speed equal to 10 was statistically insignificant across the entire pandemic. CONCLUSIONS: While COVID-19 continued to circulate in South Asia, the rate of transmission had remained below the outbreak threshold for well over a year ahead of the WHO declaration. COVID-19 is endemic in the region and no longer reaches the threshold of the pandemic definition. Both standard and enhanced surveillance metrics confirm that the pandemic had ended by the time of the WHO declaration. Prevention policies should be a focus ahead of future pandemics. On that point, policy should emphasize an epidemiological task force with widespread testing and a contact-tracing system.

COVID-19 outbreak and genomic investigation in an inpatient behavioral health unit.

Background: Inpatient behavioral health units (BHUs) had unique challenges in implementing interventions to mitigate coronavirus disease 2019 (COVID-19) transmission, in part due to socialization in BHU settings. The objective of this study was to identify the transmission routes and the efficacy of the mitigation strategies employed during a COVID-19 outbreak in an inpatient BHU during the Omicron surge from December 2021 to January 2022. Methods: An outbreak investigation was performed after identifying 2 COVID-19-positive BHU inpatients on December 16 and 20, 2021. Mitigation measures involved weekly point prevalence testing for all inpatients, healthcare workers (HCWs), and staff, followed by infection prevention mitigation measures and molecular surveillance. Whole-genome sequencing on a subset of COVID-19-positive individuals was performed to identify the outbreak source. Finally, an outbreak control sustainability plan was formulated for future BHU outbreak resurgences. Results: We identified 35 HCWs and 8 inpatients who tested positive in the BHU between December 16, 2021, and January 17, 2022. We generated severe acute respiratory coronavirus virus 2 (SARS-CoV-2) genomes from 15 HCWs and all inpatients. Phylogenetic analyses revealed 3 distinct but genetically related clusters: (1) an HCW and inpatient outbreak likely initiated by staff, (2) an HCW and inpatient outbreak likely initiated by an inpatient visitor, and (3) an HCW-only cluster initiated by staff. Conclusions: Distinct transmission clusters are consistent with multiple, independent SARS-CoV-2 introductions with further inpatient transmission occurring in communal settings. The implemented outbreak control plan comprised of enhanced personal protective equipment requirements, limited socialization, and molecular surveillance likely minimized disruptions to patient care as a model for future pandemics.

Updated Surveillance Metrics and History of the COVID-19 Pandemic (2020-2023) in the Middle East and North Africa: Longitudinal Trend Analysis.

BACKGROUND: This study updates the COVID-19 pandemic surveillance in the Middle East and North Africa (MENA) we first conducted in 2020 with 2 additional years of data for the region. OBJECTIVE: The objective of this study is to determine whether the MENA region meets the criteria for moving from a pandemic to endemic. In doing so, this study considers pandemic trends, dynamic and genomic surveillance methods, and region-specific historical context for the pandemic. These considerations continue through the World Health Organization (WHO) declaration of the end of the public health emergency for the COVID-19 pandemic on May 5, 2023. METHODS: In addition to updates to traditional surveillance data and dynamic panel estimates from the original study by Post et al, this study used data on sequenced SARS-CoV-2 variants from the Global Initiative on Sharing All Influenza Data (GISAID) to identify the appearance and duration of variants of concern. We used Nextclade nomenclature to collect clade designations from sequences and Pangolin nomenclature for lineage designations of SARS-CoV-2. Finally, we conducted a 1-sided t test to determine whether regional weekly speed of COVID-19 spread was greater than an outbreak threshold of 10. We ran the test iteratively with 6 months of data from September 4, 2020, to May 12, 2023. RESULTS: The speed of COVID-19 spread for the region had remained below the outbreak threshold for 7 continuous months by the time of the WHO declaration. Acceleration and jerk were also low and stable. Although the 1- and 7-day persistence coefficients remained statistically significant and positive, the weekly shift parameters suggested the coefficients had most recently turned negative, meaning the clustering effect of new COVID-19 cases became even smaller in the 2 weeks around the WHO declaration. From December 2021 onward, Omicron was the predominant variant of concern in sequenced viral samples. The rolling t test of the speed of spread equal to 10 became entirely insignificant from October 2022 onward. CONCLUSIONS: The COVID-19 pandemic had far-reaching effects on MENA, impacting health care systems, economies, and social well-being. Although COVID-19 continues to circulate in the MENA region, the rate of transmission remained well below the threshold of an outbreak for over 1 year ahead of the WHO declaration. COVID-19 is endemic in the region and no longer reaches the threshold of the pandemic definition. Both standard and enhanced surveillance metrics confirm that the pandemic had transitioned to endemic by the time of the WHO declaration.

Deviations in RSV epidemiological patterns and population structures in the United States following the COVID-19 pandemic.

Respiratory Syncytial Virus (RSV) is a leading cause of acute respiratory tract infection, with the greatest impact on infants, immunocompromised individuals, and older adults. RSV prevalence decreased substantially in the United States (US) following the implementation of COVID-19-related non-pharmaceutical interventions but later rebounded with abnormal seasonality. The biological and epidemiological factors underlying this altered behavior remain poorly defined. In this retrospective cohort study from 2009 to 2023 in Chicago, Illinois, US, we examined RSV epidemiology, clinical severity, and genetic diversity. We found that changes in RSV diagnostic platforms drove increased detections in outpatient settings post-2020 and that hospitalized adults infected with RSV-A were at higher risk of intensive care admission than those with RSV-B. While population structures of RSV-A remained unchanged, RSV-B exhibited a genetic shift into geographically distinct clusters. Mutations in the antigenic regions of the fusion protein suggest convergent evolution with potential implications for vaccine and therapeutic development.

Updated Surveillance Metrics and History of the COVID-19 Pandemic (2020-2023) in Latin America and the Caribbean: Longitudinal Trend Analysis.

BACKGROUND: In May 2020, the World Health Organization (WHO) declared Latin America and the Caribbean (LAC) the epicenter of the COVID-19 pandemic, with over 40% of worldwide COVID-19-related deaths at the time. This high disease burden was a result of the unique circumstances in LAC. OBJECTIVE: This study aimed to (1) measure whether the pandemic was expanding or contracting in LAC when the WHO declared the end of COVID-19 as a public health emergency of international concern on May 5, 2023; (2) use dynamic and genomic surveillance methods to describe the history of the pandemic in the region and situate the window of the WHO declaration within the broader history; and (3) provide, with a focus on prevention policies, a historical context for the course of the pandemic in the region. METHODS: In addition to updates of traditional surveillance data and dynamic panel estimates from the original study, we used data on sequenced SARS-CoV-2 variants from the Global Initiative on Sharing All Influenza Data (GISAID) to identify the appearance and duration of variants of concern (VOCs). We used Nextclade nomenclature to collect clade designations from sequences and Pangolin nomenclature for lineage designations of SARS-CoV-2. Additionally, we conducted a 1-sided t test for whether the regional weekly speed (rate of novel COVID-19 transmission) was greater than an outbreak threshold of 10. We ran the test iteratively with 6 months of data across the period from August 2020 to May 2023. RESULTS: The speed of pandemic spread for the region had remained below the outbreak threshold for 6 months by the time of the WHO declaration. Acceleration and jerk were also low and stable. Although the 1- and 7-day persistence coefficients remained statistically significant for the 120-day period ending on the week of May 5, 2023, the coefficients were relatively modest in magnitude (0.457 and 0.491, respectively). Furthermore, the shift parameters for either of the 2 most recent weeks around May 5, 2023, did not indicate any change in this clustering effect of cases on future cases. From December 2021 onward, Omicron was the predominant VOC in sequenced viral samples. The rolling t test of speed=10 became entirely insignificant from January 2023 onward. CONCLUSIONS: Although COVID-19 continues to circulate in LAC, surveillance data suggest COVID-19 is endemic in the region and no longer reaches the threshold of the pandemic definition. However, the region experienced a high COVID-19 burden in the early stages of the pandemic, and prevention policies should be an immediate focus in future pandemics. Ahead of vaccination development, these policies can include widespread testing of individuals and an epidemiological task force with a contact-tracing system.

Updated Surveillance Metrics and History of the COVID-19 Pandemic (2020-2023) in Europe: Longitudinal Trend Analysis.

BACKGROUND: In this study, we built upon our initial research published in 2020 by incorporating an additional 2 years of data for Europe. We assessed whether COVID-19 had shifted from the pandemic to endemic phase in the region when the World Health Organization (WHO) declared the end of the public health emergency of international concern on May 5, 2023. OBJECTIVE: We first aimed to measure whether there was an expansion or contraction in the pandemic in Europe at the time of the WHO declaration. Second, we used dynamic and genomic surveillance methods to describe the history of the pandemic in the region and situate the window of the WHO declaration within the broader history. Third, we provided the historical context for the course of the pandemic in Europe in terms of policy and disease burden at the country and region levels. METHODS: In addition to the updates of traditional surveillance data and dynamic panel estimates from the original study, this study used data on sequenced SARS-CoV-2 variants from the Global Initiative on Sharing All Influenza Data to identify the appearance and duration of variants of concern. We used Nextclade nomenclature to collect clade designations from sequences and Pangolin nomenclature for lineage designations of SARS-CoV-2. Finally, we conducted a 1-tailed t test for whether regional weekly speed was greater than an outbreak threshold of 10. We ran the test iteratively with 6 months of data across the sample period. RESULTS: Speed for the region had remained below the outbreak threshold for 4 months by the time of the WHO declaration. Acceleration and jerk were also low and stable. While the 1-day and 7-day persistence coefficients remained statistically significant, the coefficients were moderate in magnitude (0.404 and 0.547, respectively; P<.001 for both). The shift parameters for the 2 weeks around the WHO declaration were small and insignificant, suggesting little change in the clustering effect of cases on future cases at the time. From December 2021 onward, Omicron was the predominant variant of concern in sequenced viral samples. The rolling t test of speed equal to 10 became insignificant for the first time in April 2023. CONCLUSIONS: While COVID-19 continues to circulate in Europe, the rate of transmission remained below the threshold of an outbreak for 4 months ahead of the WHO declaration. The region had previously been in a nearly continuous state of outbreak. The more recent trend suggested that COVID-19 was endemic in the region and no longer reached the threshold of the pandemic definition. However, several countries remained in a state of outbreak, and the conclusion that COVID-19 was no longer a pandemic in Europe at the time is unclear.

Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19.

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

Genomic characterization of SARS-CoV-2 in Guinea, West Africa.

SARS-CoV-2 has claimed several million lives since its emergence in late 2019. The ongoing evolution of the virus has resulted in the periodic emergence of new viral variants with distinct fitness advantages, including enhanced transmission and immune escape. While several SARS-CoV-2 variants of concern trace their origins back to the African continent-including Beta, Eta, and Omicron-most countries in Africa remain under-sampled in global genomic surveillance efforts. In an effort to begin filling these knowledge gaps, we conducted retrospective viral genomic surveillance in Guinea from October 2020 to August 2021. We found that SARS-CoV-2 clades 20A, 20B, and 20C dominated throughout 2020 until the coincident emergence of the Alpha and Eta variants of concern in January 2021. The Alpha variant remained dominant throughout early 2021 until the arrival of the Delta variant in July. Surprisingly, despite the small sample size of our study, we also found the persistence of the early SARS-CoV-2 clade 19B as late as April 2021. Together, these data help fill in our understanding of the SARS-CoV-2 population dynamics in West Africa early in the COVID-19 pandemic.

The impact of an intervention to reduce dispersal from wastewater drain sites on carbapenem-resistant Pseudomonas aeruginosa colonization and bloodstream infection on a hematopoietic cell transplant and hematologic malignancy unit.

OBJECTIVE: To evaluate the impact of an intervention to limit dispersal from wastewater drain (WWD) sites on meropenem-nonsusceptible Pseudomonas aeruginosa patient and environmental colonization and bloodstream infection (BSI) on a hematopoietic cell transplant (HCT) and hematologic malignancy (HM) unit. DESIGN: This quasi-experimental study included pre/postintervention point-prevalence surveys in July 2019 and June 2020, respectively. The retrospective cohort included HCT/HM patients with P. aeruginosa BSI between 2012 and 2022. SETTING: Adult HCT/HM unit at an academic center. PARTICIPANTS: This study included consenting HCT/HM patients on the unit at the time of the point-prevalence surveys. HCT/HM patients with P. aeruginosa BSI between 2012 and 2022. METHODS: A quality improvement intervention targeting WWD sites was conceived and implemented on a HCT/HM unit. Pre and postintervention colonization samples were obtained from patients and environmental sites, cultivated on selective media, then characterized by susceptibility testing. Whole-genome sequencing and phylogenetic analysis were performed on select isolates. The impact of the intervention on colonization and BSI was evaluated, as was relatedness among isolates. RESULTS: Although colonization of WWD sites with meropenem-nonsusceptible P. aeruginosa was widespread before and after this intervention, we observed a substantial decline in patient colonization (prevalence rate ratio, 0.35; 95% confidence interval [CI], 0.04-3.12) and BSI (incidence rate ratio, 0.67; 95% CI, 0.31-1.42) after the intervention. Among 3 predominant sequence types (ST-111, ST-446, and ST-308), there was striking genetic conservation within groups and among environmental colonization, patient colonization, and BSI isolates. CONCLUSIONS: An intervention targeting WWD sites on a HCT/HM unit had a meaningful impact on meropenem-nonsusceptible P. aeruginosa patient colonization and BSI.

Integration of individualized and population-level molecular epidemiology data to model COVID-19 outcomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with enhanced transmissibility and immune escape have emerged periodically throughout the coronavirus disease 2019 (COVID-19) pandemic, but the impact of these variants on disease severity has remained unclear. In this single-center, retrospective cohort study, we examined the association between SARS-CoV-2 clade and patient outcome over a two-year period in Chicago, Illinois. Between March 2020 and March 2022, 14,252 residual diagnostic specimens were collected from SARS-CoV-2-positive inpatients and outpatients alongside linked clinical and demographic metadata, of which 2,114 were processed for viral whole-genome sequencing. When controlling for patient demographics and vaccination status, several viral clades were associated with risk for hospitalization, but this association was negated by the inclusion of population-level confounders, including case count, sampling bias, and shifting standards of care. These data highlight the importance of integrating non-virological factors into disease severity and outcome models for the accurate assessment of patient risk.

A synthetic biology approach to assemble and reboot clinically relevant Pseudomonas aeruginosa tailed phages.

The rise in the frequency of antibiotic resistance has made bacterial infections, specifically Pseudomonas aeruginosa, a cause for greater concern. Phage therapy is a promising solution that uses naturally isolated phages to treat bacterial infections. Ecological limitations, which stipulate a discrete host range and the inevitable evolution of resistance, may be overcome through a better understanding of phage biology and the utilization of engineered phages. In this study, we developed a synthetic biology approach to construct tailed phages that naturally target clinically relevant strains of Pseudomonas aeruginosa. As proof of concept, we successfully cloned and assembled the JG024 and DMS3 phage genomes in yeast using transformation-associated recombination cloning and rebooted these two phage genomes in two different strains of P. aeruginosa. We identified factors that affected phage reboot efficiency like the phage species or the presence of antiviral defense systems in the bacterial strain. We have successfully extended this method to two other phage species and observed that the method enables the reboot of phages that are naturally unable to infect the strain used for reboot. This research represents a critical step toward the construction of clinically relevant, engineered P. aeruginosa phages.IMPORTANCEPseudomonas aeruginosa is a bacterium responsible for severe infections and a common major complication in cystic fibrosis. The use of antibiotics to treat bacterial infections has become increasingly difficult as antibiotic resistance has become more prevalent. Phage therapy is an alternative solution that is already being used in some European countries, but its use is limited by the narrow host range due to the phage receptor specificity, the presence of antiviral defense systems in the bacterial strain, and the possible emergence of phage resistance. In this study, we demonstrate the use of a synthetic biology approach to construct and reboot clinically relevant P. aeruginosa tailed phages. This method enables a significant expansion of possibilities through the construction of engineered phages for therapy applications.

Social/Sexual Networks of People Newly Diagnosed with HIV in Ibadan, Nigeria.

Young men who have sex with men (YMSM) in Nigeria are ten times more likely to be living with HIV-1 than other young men. Due to stigma and criminalization of same-sex sexual behavior, YMSM sexual networks are likely to overlap with those of the general population, leading to a generalized HIV-1 epidemic. Due to limited research on social/sexual network dynamics related to HIV-1 in Nigeria, our study focused on YMSM and sought to assess the feasibility and acceptability of collecting social and sexual network data in Network Canvas from individuals newly diagnosed with HIV-1 in Ibadan, Nigeria. The Network Canvas software was piloted at three sites in Ibadan, Nigeria to collect social/sexual network data from 151 individuals newly diagnosed with HIV-1. Our study sample included 37.7% YMSM; participants reported a mean of 2.6 social alters and 2.6 sexual alters. From the 151 egos and 634 alters, 85 potential unique individuals (194 total) were identified; 65 egos/alters were collapsed into 25 unique individuals. Our success collecting network data from individuals newly diagnosed with HIV-1 in Ibadan demonstrates clear feasibility and acceptability of the approach and the use of Network Canvas to capture and manage these data.

Evaluation of the analytical performance of the 15-minute point-of-care DASH™ SARS-CoV-2 RT-qPCR test.

Accurate and timely diagnosis for COVID-19 diagnosis allows highly effective antiviral medications to be prescribed. The DASH™ Rapid PCR System is a sample-to-answer point-of-care platform combining state-of-the-art PCR kinetics with sequence specific hybridization. The platform's first assay, the DASH™ SARS-CoV-2/S test for anterior nares direct swab specimens, received FDA Emergency Use Authorization in March 2022 for point-of-care use. Here we report the analytical characteristics of the assay including limit of detection, dynamic range, and robustness of SARS-CoV-2 variant detection. The limit of detection was determined by testing swabs contrived with one hundred copies of wild type or Omicron BA.5 virus and detecting 20/20 and 19/20, respectively. The dynamic range was assessed with contrived swabs containing 102-106 copies; the log-linear relationship between Cq and copy input was plotted, and the qPCR efficiency calculated from the slope of the line was 101.4%. Detection of seven SARS-CoV-2 variants was demonstrated.

Phenotypes of a Pseudomonas aeruginosa hypermutator lineage that emerged during prolonged mechanical ventilation in a patient without cystic fibrosis.

Hypermutator lineages of Pseudomonas aeruginosa arise frequently during the years of airway infection experienced by patients with cystic fibrosis and bronchiectasis but are rare in the absence of chronic infection and structural lung disease. Since the onset of the COVID-19 pandemic, large numbers of patients have remained mechanically ventilated for extended periods of time. These patients are prone to acquire bacterial pathogens that persist for many weeks and have the opportunity to evolve within the pulmonary environment. However, little is known about what types of adaptations occur in these bacteria and whether these adaptations mimic those observed in chronic infections. We describe a COVID-19 patient with a secondary P. aeruginosa lung infection in whom the causative bacterium persisted for >50 days. Over the course of this infection, a hypermutator lineage of P. aeruginosa emerged and co-existed with a non-hypermutator lineage. Compared to the parental lineage, the hypermutator lineage evolved to be less cytotoxic and less virulent. Genomic analyses of the hypermutator lineage identified numerous mutations, including in the mismatch repair gene mutL and other genes frequently mutated in individuals with cystic fibrosis. Together, these findings demonstrate that hypermutator lineages can emerge when P. aeruginosa persists following acute infections such as ventilator-associated pneumonia and that these lineages have the potential to affect patient outcomes.IMPORTANCEPseudomonas aeruginosa may evolve to accumulate large numbers of mutations in the context of chronic infections such as those that occur in individuals with cystic fibrosis. However, these "hypermutator" lineages are rare following acute infections. Here, we describe a non-cystic fibrosis patient with COVID-19 pneumonia who remained mechanically ventilated for months. The patient became infected with a strain of P. aeruginosa that evolved to become a hypermutator. We demonstrate that hypermutation led to changes in cytotoxicity and virulence. These findings are important because they demonstrate that P. aeruginosa hypermutators can emerge following acute infections and that they have the potential to affect patient outcomes in this setting.

Klebsiella pneumoniae clinical isolates with features of both multidrug-resistance and hypervirulence have unexpectedly low virulence.

Klebsiella pneumoniae has been classified into two types, classical K. pneumoniae (cKP) and hypervirulent K. pneumoniae (hvKP). cKP isolates are highly diverse and important causes of nosocomial infections; they include globally disseminated antibiotic-resistant clones. hvKP isolates are sensitive to most antibiotics but are highly virulent, causing community-acquired infections in healthy individuals. The virulence phenotype of hvKP is associated with pathogenicity loci responsible for siderophore and hypermucoid capsule production. Recently, convergent strains of K. pneumoniae, which possess features of both cKP and hvKP, have emerged and are cause of much concern. Here, we screen the genomes of 2,608 multidrug-resistant K. pneumoniae isolates from the United States and identify 47 convergent isolates. We perform phenotypic and genomic characterization of 12 representative isolates. These 12 convergent isolates contain a variety of antimicrobial resistance plasmids and virulence plasmids. Most convergent isolates contain aerobactin biosynthesis genes and produce more siderophores than cKP isolates but not more capsule. Unexpectedly, only 1 of the 12 tested convergent isolates has a level of virulence consistent with hvKP isolates in a murine pneumonia model. These findings suggest that additional studies should be performed to clarify whether convergent strains are indeed more virulent than cKP in mouse and human infections.