B cell genomics behind cross-neutralization of SARS-CoV-2 variants and SARS-CoV.

Monoclonal antibodies (mAbs) are a focus in vaccine and therapeutic design to counteract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants. Here, we combined B cell sorting with single-cell VDJ and RNA sequencing (RNA-seq) and mAb structures to characterize B cell responses against SARS-CoV-2. We show that the SARS-CoV-2-specific B cell repertoire consists of transcriptionally distinct B cell populations with cells producing potently neutralizing antibodies (nAbs) localized in two clusters that resemble memory and activated B cells. Cryo-electron microscopy structures of selected nAbs from these two clusters complexed with SARS-CoV-2 spike trimers show recognition of various receptor-binding domain (RBD) epitopes. One of these mAbs, BG10-19, locks the spike trimer in a closed conformation to potently neutralize SARS-CoV-2, the recently arising mutants B.1.1.7 and B.1.351, and SARS-CoV and cross-reacts with heterologous RBDs. Together, our results characterize transcriptional differences among SARS-CoV-2-specific B cells and uncover cross-neutralizing Ab targets that will inform immunogen and therapeutic design against coronaviruses.

Telehealth Interventions for HIV in Low- and Middle-Income Countries.

PURPOSE OF REVIEW: This review summarizes technology-based interventions for HIV in low- and middle-income countries (LMICs). We highlight potential benefits and challenges to using telehealth in LMICs and propose areas for future study. RECENT FINDINGS: We identified several models for using telehealth to expand HIV health care access in LMICs, including telemedicine visits for pre-exposure prophylaxis (PrEP) and antiretroviral therapy (ART) services, telementoring programs for providers, and virtual peer-support groups. Emerging data support the acceptability and feasibility of these strategies. However, further investigations are needed to determine whether these models are scalable and sustainable in the face of barriers related to cost, infrastructure, and regulatory approval. HIV telehealth interventions may be a valuable approach to addressing gaps along the HIV care cascade in LMICs. Future studies should focus on strategies for expanding existing programs to scale and for assessing long-term clinical outcomes.

Multi-institute analysis of carbapenem resistance reveals remarkable diversity, unexplained mechanisms, and limited clonal outbreaks.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by blaKPC2, blaKPC3, and blaKPC4, which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.

Integrated clinical and quality improvement coaching in Son La Province, Vietnam: a model of building public sector capacity for sustainable HIV care delivery.

BACKGROUND: The global scale-up of antiretroviral therapy included extensive training and onsite support to build the capacity of HIV health care workers. However, traditional efforts aimed at strengthening knowledge and skills often are not successful at improving gaps in the key health systems required for sustaining high quality care. METHODS: We trained and mentored existing staff of the Son La provincial health department and provincial HIV clinic to work as a provincial coaching team (PCT) to provide integrated coaching in clinical HIV skills and quality improvement (QI) to the HIV clinics in the province. Nine core indicators were measured through chart extraction by clinic and provincial staff at baseline and at 6 month intervals thereafter. Coaching from the team to each of the clinics, in both QI and clinical skills, was guided by results of performance measurements, gap analyses, and resulting QI plans. RESULTS: After 18 months, the PCT had successfully spread QI activities, and was independently providing regular coaching to the provincial general hospital clinic and six of the eight district clinics in the province. The frequency and type of coaching was determined by performance measurement results. Clinics completed a mean of five QI projects. Quality of HIV care was improved throughout all clinics with significant increases in seven of the indicators. Overall both the PCT activities and clinic performance were sustained after integration of the model into the Vietnam National QI Program. CONCLUSIONS: We successfully built capacity of a team of public sector health care workers to provide integrated coaching in both clinical skills and QI across a province. The PCT is a feasible and effective model to spread and sustain quality activities and improve HIV care services in a decentralized rural setting.

Staphylococcal enterotoxin P predicts bacteremia in hospitalized patients colonized with methicillin-resistant Staphylococcus aureus.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) colonization predicts later infection, with both host and pathogen determinants of invasive disease. METHODS: This nested case-control study evaluates predictors of MRSA bacteremia in an 8-intensive care unit (ICU) prospective adult cohort from 1 September 2003 through 30 April 2005 with active MRSA surveillance and collection of ICU, post-ICU, and readmission MRSA isolates. We selected MRSA carriers who did (cases) and those who did not (controls) develop MRSA bacteremia. Generating assembled genome sequences, we evaluated 30 MRSA genes potentially associated with virulence and invasion. Using multivariable Cox proportional hazards regression, we assessed the association of these genes with MRSA bacteremia, controlling for host risk factors. RESULTS: We collected 1578 MRSA isolates from 520 patients. We analyzed host and pathogen factors for 33 cases and 121 controls. Predictors of MRSA bacteremia included a diagnosis of cancer, presence of a central venous catheter, hyperglycemia (glucose level, >200 mg/dL), and infection with a MRSA strain carrying the gene for staphylococcal enterotoxin P (sep). Receipt of an anti-MRSA medication had a significant protective effect. CONCLUSIONS: In an analysis controlling for host factors, colonization with MRSA carrying sep increased the risk of MRSA bacteremia. Identification of risk-adjusted genetic determinants of virulence may help to improve prediction of invasive disease and suggest new targets for therapeutic intervention.

At-home Testing and Risk Factors for Acquisition of SARS-CoV-2 Infection in a Major US Metropolitan Area.

IMPORTANCE: Unbiased assessment of risks associated with acquisition of SARS-CoV-2 is critical to informing mitigation efforts during pandemics. OBJECTIVE: Understand risk factors for acquiring COVID-19 in a large, prospective cohort of adult residents recruited to be representative of a large US metropolitan area. DESIGN: Fully remote longitudinal cohort study launched in October 2020 and ongoing; Study data reported through June 15, 2021. SETTING: Brigham and Women’s Hospital, Boston MA. PARTICIPANTS: Adults within 45 miles of Boston, MA. INTERVENTION: Monthly at-home SARS-CoV-2 viral and antibody testing. MAIN OUTCOMES: Between October 2020 and January 2021, we enrolled 10,289 adults reflective of Massachusetts census data. At study entry, 567 (5.5%) participants had evidence of current or prior SARS-CoV-2 infection. This increased to 13.4% by June 15, 2021. Compared to whites, Black non-Hispanic participants had a 2.2 fold greater risk of acquiring COVID-19 (HR 2.19, 95% CI 1.91-2.50; p=<0.001) and Hispanics had a 1.5 fold greater risk (HR 1.52, 95% CI 1.32-1.71; p=<0.016). Individuals aged 18-29, those who worked outside the home, and those living with other adults and children were at an increased risk. Individuals in the second and third lowest disadvantaged neighborhood communities, as measured by the area deprivation index as a marker for socioeconomic status by census block group, were associated with an increased risk in developing COVID-19. Individuals with medical risk factors for severe COVID-19 disease were at a decreased risk of SARS-CoV-2 acquisition. CONCLUSIONS: These results demonstrate that race/ethnicity and socioeconomic status are not only risk factors for severity of disease but are also the biggest determinants of acquisition of infection. Importantly, this disparity is significantly underestimated if based on PCR data alone as noted by the discrepancy in serology vs. PCR detection for non-white participants, and points to persistent disparity in access to testing. Meanwhile, medical conditions and advanced age that increase the risk for severity of SARS-CoV-2 disease were associated with a lower risk of acquisition of COVID-19 suggesting the importance of behavior modifications. These findings highlight the need for mitigation programs that overcome challenges of structural racism in current and future pandemics. TRIAL REGISTRATION: N/A. QUESTION: What population and occupational groups in the United States are at increased risk for acquiring COVID-19? FINDINGS: In this remote, longitudinal cohort study involving monthly PCR and serology self-testing of 10,289 adult residents of the Boston metropolitan area, 9257 (90.0%) of TestBoston participants acquired evidence of immunity to SARS-CoV-2 through vaccination, infection, or both as of June 15, 2021. Residents identifying as Black, Hispanic/Latinx had an increased risk of acquisition of COVID-19. Healthcare workers were not at increased risk of SARS-CoV-2 acquisition. Individuals with medical risk factors for severe COVID-19 disease were at a decreased risk of SARS-CoV-2 acquisition. MEANING: These results demonstrate that race/ethnicity and socioeconomic status are not only risk factors for severity of disease but also are the biggest determinants of acquisition of infection. These findings highlight the need to address the consequences of structural racism during the development of mitigation programs for current and future pandemics.

Undergraduate Medical Education Reform in Viet Nam for a Primary Health Care Workforce.

Strong primary health care (PHC) systems require a robust PHC workforce. Traditionally, medical education takes place in academic medical centres that favour subspecialty care rather than PHC settings. This may undervalue primary care as a career and contribute to a shortage of PHC workers. However, designing undergraduate medical education curricula that incorporate early experiences in clinical care delivery at PHC sites remains a challenge, including in many low- and middle-income countries (LMICs). This paper describes how a collaboration between Harvard Medical School and five medical schools in Vietnam, and in-country collaborations among the Vietnamese medical schools, facilitated curricular innovation and co-creation of coursework relevant to PHC through the development of a Practice of Medicine (POM) course. The collaboration implemented a technical assistance strategy consisting of in-person workshops, focused virtual consultations, on-site 'office hours', site visits and observations to each of the five medical universities, and immersion trips to support the creation and implementation of the POM course. A pilot program was started at a single site and then scaled nationally using local customisation, experience, and expertise utilising a train-the-trainers approach. As a result, five new POM courses have been developed by five Vietnamese institutions. Fifty Vietnamese faculty received training to lead the POM course development, and 228 community-based preceptors have been trained to teach students at PHC sites. A total of 52 new PHC and community-based clinical training sites have been added, and 3,615 students have completed or are currently going through a POM course. This experience can serve as a model for future academic collaborations to support the development of a robust PHC workforce for the 21st century.

At-home Testing and Risk Factors for Acquisition of SARS-CoV-2 Infection in a Major US Metropolitan Area.

Background: Unbiased assessment of the risks associated with acquisition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to informing mitigation efforts during pandemics. The objective of our study was to understand the risk factors for acquiring coronavirus disease 2019 (COVID-19) in a large prospective cohort of adult residents in a large US metropolitan area. Methods: We designed a fully remote longitudinal cohort study involving monthly at-home SARS-CoV-2 polymerase chain reaction (PCR) and serology self-testing and monthly surveys. Results: Between October 2020 and January 2021, we enrolled 10 289 adults reflective of the Boston metropolitan area census data. At study entry, 567 (5.5%) participants had evidence of current or prior SARS-CoV-2 infection. This increased to 13.4% by June 15, 2021. Compared with Whites, Black non-Hispanic participants had a 2.2-fold greater risk of acquiring COVID-19 (hazard ratio [HR], 2.19; 95% CI, 1.91-2.50; P < .001), and Hispanics had a 1.5-fold greater risk (HR, 1.52; 95% CI, 1.32-1.71; P < .016). Individuals aged 18-29, those who worked outside the home, and those living with other adults and children were at an increased risk. Individuals in the second and third lowest disadvantaged neighborhood communities were associated with an increased risk of acquiring COVID-19. Individuals with medical risk factors for severe disease were at a decreased risk of SARS-CoV-2 acquisition. Conclusions: These results demonstrate that race/ethnicity and socioeconomic status are the biggest determinants of acquisition of infection. This disparity is significantly underestimated if based on PCR data alone, as noted by the discrepancy in serology vs PCR detection for non-White participants, and points to persistent disparity in access to testing. Medical conditions and advanced age, which increase the risk for severity of SARS-CoV-2 disease, were associated with a lower risk of COVID-19 acquisition, suggesting the importance of behavior modifications. These findings highlight the need for mitigation programs that overcome challenges of structural racism in current and future pandemics.

A pretest-posttest design to assess the effectiveness of an intervention to reduce HIV-related stigma and discrimination in healthcare settings in Vietnam.

INTRODUCTION: Stigma and discrimination are important barriers to HIV epidemic control. We implemented a multi-pronged facility-level intervention to reduce stigma and discrimination at health facilities across three high-burden provinces. Key components of the intervention included measurement of stigma, data review and use, participatory training of healthcare workers (HCWs), and engagement of people living with HIV and key populations in all stigma reduction activities. METHODS: From July 2018 to July 2019, we assessed HIV-related stigma and discrimination among patients and HCWs at 10 facilities at baseline and 9 months following an intervention. A repeated measures design was used to assess the change in stigma and discrimination among HCWs and a repeated cross-sectional design assessed the change in stigma and discrimination experienced by PLHIV. HCWs at target facilities were invited at random and PLHIV were recruited when presenting for care during the two assessment periods. McNemar's test was used to compare paired proportions among HCWs, and chi-square test was used to compare proportions among PLHIV. Mixed models were used to compare outcomes before and after the intervention. RESULTS: Semi-structured interviews were conducted with 649 and 652 PLHIV prior to and following the intervention, respectively. At baseline, over the previous 12 months, 21% reported experiencing discrimination, 16% reported self-stigma, 14% reported HIV disclosure without consent and 7% had received discriminatory reproductive health advice. Nine months after the intervention, there was a decrease in reported stigma and discrimination across all domains to 15%, 11%, 7% and 3.5%, respectively (all p-values <0.05). Among HCWs, 672 completed the pre- and post-intervention assessment. At baseline, 81% reported fear of HIV infection, 69% reported using unnecessary precautions when caring for PLHIV, 44% reported having observed other staff discriminate against PLHIV, 54% reported negative attitudes towards PLHIV and 41% felt uncomfortable working with colleagues living with HIV. The proportions decreased after the intervention to 52%, 34%, 32%, 35% and 24%, respectively (all p-values <0.05). CONCLUSIONS: A multi-pronged facility-level intervention was successful at reducing healthcare-associated HIV-related stigma in Vietnam. The findings support the scale-up of this intervention in Vietnam and highlight key components potentially applicable in other settings.

Feasibility and lessons learned on remote trial implementation from TestBoston, a fully remote, longitudinal, large-scale COVID-19 surveillance study.

Longitudinal clinical studies traditionally require in-person study visits which are well documented to pose barriers to participation and contribute challenges to enrolling representative samples. Remote trial models may reduce barriers to research engagement, improve retention, and reach a more representative cohort. As remote trials become more common following the COVID-19 pandemic, a critical evaluation of this approach is imperative to optimize this paradigm shift in research. The TestBoston study was launched to understand prevalence and risk factors for COVID-19 infection in the greater Boston area through a fully remote home-testing model. Participants (adults, within 45 miles of Boston, MA) were recruited remotely from patient registries at Brigham and Women's Hospital and the general public. Participants were provided with monthly and "on-demand" at-home SARS-CoV-2 RT-PCR and antibody testing using nasal swab and dried blood spot self-collection kits and electronic surveys to assess symptoms and risk factors for COVID-19 via an online dashboard. Between October 2020 and January 2021, we enrolled 10,289 participants reflective of Massachusetts census data. Mean age was 47 years (range 18-93), 5855 (56.9%) were assigned female sex at birth, 7181(69.8%) reported being White non-Hispanic, 952 (9.3%) Hispanic/Latinx, 925 (9.0%) Black, 889 (8.6%) Asian, and 342 (3.3%) other and/or more than one race. Lower initial enrollment among Black and Hispanic/Latinx individuals required an adaptive approach to recruitment, leveraging connections to the medical system, coupled with community partnerships to ensure a representative cohort. Longitudinal retention was higher among participants who were White non-Hispanic, older, working remotely, and with lower socioeconomic vulnerability. Implementation highlighted key differences in remote trial models as participants independently navigate study milestones, requiring a dedicated participant support team and robust technology platforms, to reduce barriers to enrollment, promote retention, and ensure scientific rigor and data quality. Remote clinical trial models offer tremendous potential to engage representative cohorts, scale biomedical research, and promote accessibility by reducing barriers common in traditional trial design. Barriers and burdens within remote trials may be experienced disproportionately across demographic groups. To maximize engagement and retention, researchers should prioritize intensive participant support, investment in technologic infrastructure and an adaptive approach to maximize engagement and retention.

Inter-species geographic signatures for tracing horizontal gene transfer and long-term persistence of carbapenem resistance.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE) are an urgent global health threat. Inferring the dynamics of local CRE dissemination is currently limited by our inability to confidently trace the spread of resistance determinants to unrelated bacterial hosts. Whole-genome sequence comparison is useful for identifying CRE clonal transmission and outbreaks, but high-frequency horizontal gene transfer (HGT) of carbapenem resistance genes and subsequent genome rearrangement complicate tracing the local persistence and mobilization of these genes across organisms. METHODS: To overcome this limitation, we developed a new approach to identify recent HGT of large, near-identical plasmid segments across species boundaries, which also allowed us to overcome technical challenges with genome assembly. We applied this to complete and near-complete genome assemblies to examine the local spread of CRE in a systematic, prospective collection of all CRE, as well as time- and species-matched carbapenem-susceptible Enterobacterales, isolated from patients from four US hospitals over nearly 5 years. RESULTS: Our CRE collection comprised a diverse range of species, lineages, and carbapenem resistance mechanisms, many of which were encoded on a variety of promiscuous plasmid types. We found and quantified rearrangement, persistence, and repeated transfer of plasmid segments, including those harboring carbapenemases, between organisms over multiple years. Some plasmid segments were found to be strongly associated with specific locales, thus representing geographic signatures that make it possible to trace recent and localized HGT events. Functional analysis of these signatures revealed genes commonly found in plasmids of nosocomial pathogens, such as functions required for plasmid retention and spread, as well survival against a variety of antibiotic and antiseptics common to the hospital environment. CONCLUSIONS: Collectively, the framework we developed provides a clearer, high-resolution picture of the epidemiology of antibiotic resistance importation, spread, and persistence in patients and healthcare networks.

Gordonia bronchialis bacteremia and pleural infection: case report and review of the literature.

Gordonia species are aerobic actinomycetes recently recognized as causing human disease, often in the setting of intravascular catheter-related infections. We describe a case of Gordonia bronchialis bacteremia and pleural space infection in the absence of an indwelling intravascular catheter and review the breadth of reported infections with this emerging pathogen.

Plasma from patients with bacterial sepsis or severe COVID-19 induces suppressive myeloid cell production from hematopoietic progenitors in vitro.

Bacterial sepsis and severe COVID-19 share similar clinical manifestations and are both associated with dysregulation of the myeloid cell compartment. We previously reported an expanded CD14+ monocyte state, MS1, in patients with bacterial sepsis and validated expansion of this cell subpopulation in publicly available transcriptomics data. Here, using published datasets, we show that the gene expression program associated with MS1 correlated with sepsis severity and was up-regulated in monocytes from patients with severe COVID-19. To examine the ontogeny and function of MS1 cells, we developed a cellular model for inducing CD14+ MS1 monocytes from healthy bone marrow hematopoietic stem and progenitor cells (HSPCs). We found that plasma from patients with bacterial sepsis or COVID-19 induced myelopoiesis in HSPCs in vitro and expression of the MS1 gene program in monocytes and neutrophils that differentiated from these HSPCs. Furthermore, we found that plasma concentrations of IL-6, and to a lesser extent IL-10, correlated with increased myeloid cell output from HSPCs in vitro and enhanced expression of the MS1 gene program. We validated the requirement for these two cytokines to induce the MS1 gene program through CRISPR-Cas9 editing of their receptors in HSPCs. Using this cellular model system, we demonstrated that induced MS1 cells were broadly immunosuppressive and showed decreased responsiveness to stimulation with a synthetic RNA analog. Our in vitro study suggests a potential role for systemic cytokines in inducing myelopoiesis during severe bacterial or SARS-CoV-2 infection.

Adaptive evolution of virulence and persistence in carbapenem-resistant Klebsiella pneumoniae.

Among the most urgent public health threats is the worldwide emergence of carbapenem-resistant Enterobacteriaceae1-4, which are resistant to the antibiotic class of 'last resort'. In the United States and Europe, carbapenem-resistant strains of the Klebsiella pneumoniae ST258 (ref. 5) sequence type are dominant, endemic6-8 and associated with high mortality6,9,10. We report the global evolution of pathogenicity in carbapenem-resistant K. pneumoniae, resulting in the repeated convergence of virulence and carbapenem resistance in the United States and Europe, dating back to as early as 2009. We demonstrate that K. pneumoniae can enhance its pathogenicity by adopting two opposing infection programs through easily acquired gain- and loss-of-function mutations. Single-nucleotide polymorphisms in the capsule biosynthesis gene wzc lead to hypercapsule production, which confers phagocytosis resistance, enhanced dissemination and increased mortality in animal models. In contrast, mutations disrupting capsule biosynthesis genes impair capsule production, which enhances epithelial cell invasion, in vitro biofilm formation and persistence in urinary tract infections. These two types of capsule mutants have emerged repeatedly and independently in Europe and the United States, with hypercapsule mutants associated with bloodstream infections and capsule-deficient mutants associated with urinary tract infections. In the latter case, drug-tolerant K. pneumoniae can persist to yield potentially untreatable, persistent infection.

Author Correction: Adaptive evolution of virulence and persistence in carbapenem-resistant Klebsiella pneumoniae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Induction of a regulatory myeloid program in bacterial sepsis and severe COVID-19.

A recent estimate suggests that one in five deaths globally are associated with sepsis 1 . To date, no targeted treatment is available for this syndrome, likely due to substantial patient heterogeneity 2,3 and our lack of insight into sepsis immunopathology 4 . These issues are highlighted by the current COVID-19 pandemic, wherein many clinical manifestations of severe SARS-CoV-2 infection parallel bacterial sepsis 5-8 . We previously reported an expanded CD14+ monocyte state, MS1, in patients with bacterial sepsis or non-infectious critical illness, and validated its expansion in sepsis across thousands of patients using public transcriptomic data 9 . Despite its marked expansion in the circulation of bacterial sepsis patients, its relevance to viral sepsis and association with disease outcomes have not been examined. In addition, the ontogeny and function of this monocyte state remain poorly characterized. Using public transcriptomic data, we show that the expression of the MS1 program is associated with sepsis mortality and is up-regulated in monocytes from patients with severe COVID-19. We found that blood plasma from bacterial sepsis or COVID-19 patients with severe disease induces emergency myelopoiesis and expression of the MS1 program, which are dependent on the cytokines IL-6 and IL-10. Finally, we demonstrate that MS1 cells are broadly immunosuppressive, similar to monocytic myeloid-derived suppressor cells (MDSCs), and have decreased responsiveness to stimulation. Our findings highlight the utility of regulatory myeloid cells in sepsis prognosis, and the role of systemic cytokines in inducing emergency myelopoiesis during severe bacterial and SARS-CoV-2 infections.

Rapid identification and phylogenetic classification of diverse bacterial pathogens in a multiplexed hybridization assay targeting ribosomal RNA.

Rapid bacterial identification remains a critical challenge in infectious disease diagnostics. We developed a novel molecular approach to detect and identify a wide diversity of bacterial pathogens in a single, simple assay, exploiting the conservation, abundance, and rich phylogenetic content of ribosomal RNA in a rapid fluorescent hybridization assay that requires no amplification or enzymology. Of 117 isolates from 64 species across 4 phyla, this assay identified bacteria with >89% accuracy at the species level and 100% accuracy at the family level, enabling all critical clinical distinctions. In pilot studies on primary clinical specimens, including sputum, blood cultures, and pus, bacteria from 5 different phyla were identified.

Simultaneous detection of genotype and phenotype enables rapid and accurate antibiotic susceptibility determination.

Multidrug resistant organisms are a serious threat to human health1,2. Fast, accurate antibiotic susceptibility testing (AST) is a critical need in addressing escalating antibiotic resistance, since delays in identifying multidrug resistant organisms increase mortality3,4 and use of broad-spectrum antibiotics, further selecting for resistant organisms. Yet current growth-based AST assays, such as broth microdilution5, require several days before informing key clinical decisions. Rapid AST would transform the care of patients with infection while ensuring that our antibiotic arsenal is deployed as efficiently as possible. Growth-based assays are fundamentally constrained in speed by doubling time of the pathogen, and genotypic assays are limited by the ever-growing diversity and complexity of bacterial antibiotic resistance mechanisms. Here we describe a rapid assay for combined genotypic and phenotypic AST through RNA detection, GoPhAST-R, that classifies strains with 94-99% accuracy by coupling machine learning analysis of early antibiotic-induced transcriptional changes with simultaneous detection of key genetic resistance determinants to increase accuracy of resistance detection, facilitate molecular epidemiology and enable early detection of emerging resistance mechanisms. This two-pronged approach provides phenotypic AST 24-36 h faster than standard workflows, with <4 h assay time on a pilot instrument for hybridization-based multiplexed RNA detection implemented directly from positive blood cultures.

Metagenomic Sequencing of an Echovirus 30 Genome From Cerebrospinal Fluid of a Patient With Aseptic Meningitis and Orchitis.

Enteroviruses cause a wide spectrum of clinical disease. In this study, we describe the case of a young man with orchitis and aseptic meningitis who was diagnosed with enterovirus infection. Using unbiased "metagenomic" massively parallel sequencing, we assembled a near-complete viral genome, the first use of this method for full-genome viral sequencing from cerebrospinal fluid. We found that the genome belonged to the subgroup echovirus 30, which is a common cause of aseptic meningitis but has not been previously reported to cause orchitis.