Multiplex detection and identification of viral, bacterial, and protozoan pathogens in human blood and plasma using an expanded high-density resequencing microarray platform.

Introduction: Nucleic acid tests for blood donor screening have improved the safety of the blood supply; however, increasing numbers of emerging pathogen tests are burdensome. Multiplex testing platforms are a potential solution. Methods: The Blood Borne Pathogen Resequencing Microarray Expanded (BBP-RMAv.2) can perform multiplex detection and identification of 80 viruses, bacteria and parasites. This study evaluated pathogen detection in human blood or plasma. Samples spiked with selected pathogens, each with one of 6 viruses, 2 bacteria and 5 protozoans were tested on this platform. The nucleic acids were extracted, amplified using multiplexed sets of primers, and hybridized to a microarray. The reported sequences were aligned to a database to identify the pathogen. To directly compare the microarray to an emerging molecular approach, the amplified nucleic acids were also submitted to nanopore next generation sequencing (NGS). Results: The BBP-RMAv.2 detected viral pathogens at a concentration as low as 100 copies/ml and a range of concentrations from 1,000 to 100,000 copies/ml for all the spiked pathogens. Coded specimens were identified correctly demonstrating the effectiveness of the platform. The nanopore sequencing correctly identified most samples and the results of the two platforms were compared. Discussion: These results indicated that the BBP-RMAv.2 could be employed for multiplex detection with potential for use in blood safety or disease diagnosis. The NGS was nearly as effective at identifying pathogens in blood and performed better than BBP-RMAv.2 at identifying pathogen-negative samples.

Detection of antibiotic resistant Enterobacterales in commercial raw pet food: a preliminary study.

Introduction: Raw diets have become popular in companion animal nutrition, but these diets may be contaminated with harmful bacteria because heat processing is not utilized to mitigate pathogens during the production process. We analyzed 24 commercially available frozen raw canine and feline diets for extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E). Methods: Samples were incubated in tryptic soy broth augmented with 50 µg/mL ampicillin to enrich for ESBL-E. ESBL-E were isolated using CHROMagar ESBL plates and isolate identification and antibiotic susceptibility testing were confirmed using the VITEK®2 instrument. Results: ESBL-E were isolated from 42% (10/24) of raw diets, with E. coli, Enterobacter cloacae complex and Klebsiella pneumoniae predominating. Most ESBL-E isolates (71%, 32/45) were multidrug-resistant. Direct plating of samples onto tryptic soy agar yielded bacterial counts >6 log10 for 2 samples from two different manufacturers. Conclusion: This preliminary study justifies further investigation into the potential contribution of raw diets to the dissemination of antibiotic resistant bacteria in companion animals and domestic living spaces.

Contributing Factors to Delays in COVID-19-Related Hospitalization Among Latinx and Spanish-Speaking Patients/Factores Que Contribuyen a Retrasos en Hospitalizaciones Relacionadas con COVID-19 Entre Pacientes Latinos e Hispano-Hablantes.

BACKGROUND: Latinx populations have been more heavily impacted by the COVID-19 pandemic than the general population of the US, including higher rates of hospitalization due to COVID-19 in eastern Massachusetts. We conducted a qualitative study to better understand the experiences of Latinx and Spanish-speaking patients who had clinically significant COVID-19 in the early months of the pandemic. METHODS: Thirteen qualitative, semistructured, phone interviews were conducted between December 2020 and April 2021 with Latinx and Spanish-speaking patients who had experienced clinically significant COVID-19 in the metro-north Boston area. Interviews were recorded and transcribed in their original languages. An a priori code tree was developed which was later iteratively revised based on emerging themes. Transcripts were thematically analyzed. RESULTS: Participants discussed their overall experiences contracting the COVID-19 infection, as well as their experiences with the disease and with being hospitalized and the months after in recovery. Family and social networks were a common support, both emotional and financial. Although they survived the disease, hospitalization had serious impacts on the mental and physical health of participants, including the remnants of trauma from hospitalization itself. IMPLICATIONS: Latinx and Spanish-speaking patients in eastern Massachusetts had specific experiences in the early months of the COVID-19 pandemic that were shaped by their living conditions and culture. It is important for health care professionals to understand these experiences so that they can design appropriate medical interventions as well as target outreach efforts that are culturally appropriate. Finally, serious attention should be paid to the mental health-related consequences of hospitalization and policies that can alleviate them.

Correction: Tracking ebolavirus genomic drift with a resequencing microarray.

[This corrects the article DOI: 10.1371/journal.pone.0263732.].

Storage and Algal Association of Bacteria That Protect Microchloropsis salina from Grazing by Brachionus plicatilis.

Loss of algal production from the crashes of algal mass cultivation systems represents a significant barrier to the economic production of microalgal-based biofuels. Current strategies for crash prevention can be too costly to apply broadly as prophylaxis. Bacteria are ubiquitous in microalgal mass production cultures, however few studies investigate their role and possible significance in this particular environment. Previously, we demonstrated the success of selected protective bacterial communities to save Microchloropsis salina cultures from grazing by the rotifer Brachionus plicatilis. In the current study, these protective bacterial communities were further characterized by fractionation into rotifer-associated, algal-associated, and free-floating bacterial fractions. Small subunit ribosomal RNA amplicon sequencing was used to identify the bacterial genera present in each of the fractions. Here, we show that Marinobacter, Ruegeria, and Boseongicola in algae and rotifer fractions from rotifer-infected cultures likely play key roles in protecting algae from rotifers. Several other identified taxa likely play lesser roles in protective capability. The identification of bacterial community members demonstrating protective qualities will allow for the rational design of microbial communities grown in stable co-cultures with algal production strains in mass cultivation systems. Such a system would reduce the frequency of culture crashes and represent an essentially zero-cost form of algal crop protection.

Tracking ebolavirus genomic drift with a resequencing microarray.

Filoviruses are emerging pathogens that cause acute fever with high fatality rate and present a global public health threat. During the 2013-2016 Ebola virus outbreak, genome sequencing allowed the study of virus evolution, mutations affecting pathogenicity and infectivity, and tracing the viral spread. In 2018, early sequence identification of the Ebolavirus as EBOV in the Democratic Republic of the Congo supported the use of an Ebola virus vaccine. However, field-deployable sequencing methods are needed to enable a rapid public health response. Resequencing microarrays (RMA) are a targeted method to obtain genomic sequence on clinical specimens rapidly, and sensitively, overcoming the need for extensive bioinformatic analysis. This study presents the design and initial evaluation of an ebolavirus resequencing microarray (Ebolavirus-RMA) system for sequencing the major genomic regions of four Ebolaviruses that cause disease in humans. The design of the Ebolavirus-RMA system is described and evaluated by sequencing repository samples of three Ebolaviruses and two EBOV variants. The ability of the system to identify genetic drift in a replicating virus was achieved by sequencing the ebolavirus glycoprotein gene in a recombinant virus cultured under pressure from a neutralizing antibody. Comparison of the Ebolavirus-RMA results to the Genbank database sequence file with the accession number given for the source RNA and Ebolavirus-RMA results compared to Next Generation Sequence results of the same RNA samples showed up to 99% agreement.

The use of laser-based diagnostics for the rapid identification of blood borne viruses in human plasma samples.

AIMS: To demonstrate the use of a laser-based method of detection as a potential diagnostic test for the rapid identification of blood borne viruses in human plasma. METHODS AND RESULTS: In this study, using light emissions from laser sparks on plasma samples, the successful differentiation of both human immunodeficiency virus (HIV) and hepatitis C virus (HCV) in both residual de-identified plasma samples and plasma samples spiked to clinically relevant levels with each virus were demonstrated using plasma from more than 20 individuals spanning six different blood types (O+, O-, A+, A-, B+, B-). CONCLUSIONS: These experiments demonstrate that mathematical analysis of spectral data from laser sparks can provide accurate results within minutes. This capability was demonstrated using both spiked laboratory plasma samples and clinical plasma samples collected from infected and uninfected individuals. SIGNIFICANCE AND IMPACT OF THE STUDY: There is an ongoing need to rapidly detect viral infections and to screen for multiple viral infections. A laser-based approach can achieve sensitive, multiplex detection with minimal sample preparation and provide results within minutes. These properties along with the flexibility to add new agent detection by adjusting the detection programming make it a promising tool for clinical diagnosis. The potential for a laser-based approach has been previously demonstrated using pathogens spiked into human blood to clinically relevant levels. This study demonstrates this same ability to detect infections in clinical and laboratory spiked plasma samples. The ability to differentiate between plasma samples from infected and uninfected donors and determine the virus type using a laser-based diagnostic has not been previously demonstrated. Furthermore, this study is the first demonstration of the capability to differentiate viral infections in clinical plasma samples whereas previously published work used laboratory samples spiked with a virus or dealt with the detection of cancer in clinical plasma samples.

Abiotic and Biotic Damage of Microalgae Generate Different Volatile Organic Compounds (VOCs) for Early Diagnosis of Algal Cultures for Biofuel Production.

Open microalgal ponds used in industrial biomass production are susceptible to a number of biotic and abiotic environmental stressors (e.g., grazers, pathogens, pH, temperature, etc.) resulting in pond crashes with high economic costs. Identification of signature chemicals to aid in rapid, non-invasive, and accurate identification of the stressors would facilitate targeted and effective treatment to save the algal crop from a catastrophic crash. Specifically, we were interested in identifying volatile organic compounds (VOCs) that can be used to as an early diagnostic for algal crop damage. Cultures of Microchloropsis gaditana were subjected to two forms of algal crop damage: (1) active grazing by the marine rotifer, Brachionus plicatilis, or (2) repeated freeze-thaw cycles. VOCs emitted above the headspace of these algal cultures were collected using fieldable solid phase microextraction (SPME) fibers. An untargeted analysis and identification of VOCs was conducted using gas chromatography-mass spectrometry (GC-MS). Diagnostic VOCs unique to each algal crop damage mechanism were identified. Active rotifer grazing of M. gaditana was characterized by the appearance of carotenoid degradation products, including ß-cyclocitral and various alkenes. Freeze-thaw algae produced a different set of VOCs, including palmitoleic acid. Both rotifer grazing and freeze-thawed algae produced ß-ionone as a VOC, possibly suggesting a common stress-induced cellular mechanism. Importantly, these identified VOCs were all absent from healthy algal cultures of M. gaditana. Early detection of biotic or abiotic environmental stressors will facilitate early diagnosis and application of targeted treatments to prevent algal pond crashes. Thus, our work further supports the use of VOCs for monitoring the health of algal ponds to ultimately enhance algal crop yields for production of biofuel.

HLA-DQA1*03:03:01:16Q, a novel allele with an acceptor splice site mutation.

DQA1*03:03:01:16Q differs from DQA1*03:03:01:01 by one nucleotide at the Intron 3 splicing acceptor site.

Acylation as a successful derivatization strategy for the analysis of pinacolyl alcohol in a glycerol-rich matrix by GC-MS: application during an OPCW Proficiency Test.

A derivatization protocol based on the acylation of pinacolyl alcohol (PA), an important marker for the nerve agent soman, is presented. The procedure provides a convenient means of detecting, by gas chromatography-mass spectrometry (GC-MS), PA when present at a low concentration in a complex glycerol/alcohol-rich matrix. While there are only two reports describing the specific analysis of PA in matrices at low concentrations, the protocol described herein represents the first of its kind in the analysis of PA in a highly reactive matrix. Two alternative paths for the protocol's execution are presented. The first involves the direct derivatization of the PA with either acetyl or benzoyl chloride; both reactions yield ester products with significantly different retention times than those of the interferences of the reactive glycerol-rich matrix and in areas of the GC-chromatogram featuring lower levels of matrix interferences. A second procedure involved an initial diethyl ether/aqueous extraction of the matrix; while the extraction was found to substantially remove many of the hydrophilic matrix components and improve the overall derivatization, it also led to some loss of PA available for the derivatization. Both protocols were applied to the successful derivatization and analysis of PA by GC-MS when present at a 5 µg.mL-1 concentration in a glycerol-rich matrix sample administered during the 48th Proficiency Test administered by the Organisation for the Prohibition of Chemical Weapons (OPCW).

Low Molecular Weight Volatile Organic Compounds Indicate Grazing by the Marine Rotifer Brachionus plicatilis on the Microalgae Microchloropsis salina.

Microalgae produce specific chemicals indicative of stress and/or death. The aim of this study was to perform non-destructive monitoring of algal culture systems, in the presence and absence of grazers, to identify potential biomarkers of incipient pond crashes. Here, we report ten volatile organic compounds (VOCs) that are robustly generated by the marine alga, Microchloropsis salina, in the presence and/or absence of the marine grazer, Brachionus plicatilis. We cultured M. salina with and without B. plicatilis and collected in situ volatile headspace samples using thermal desorption tubes over the course of several days. Data from four experiments were aggregated, deconvoluted, and chromatographically aligned to determine VOCs with tentative identifications made via mass spectral library matching. VOCs generated by algae in the presence of actively grazing rotifers were confirmed via pure analytical standards to be pentane, 3-pentanone, 3-methylhexane, and 2-methylfuran. Six other VOCs were less specifically associated with grazing but were still commonly observed between the four replicate experiments. Through this work, we identified four biomarkers of rotifer grazing that indicate algal stress/death. This will aid machine learning algorithms to chemically define and diagnose algal mass production cultures and save algae cultures from imminent crash to make biofuel an alternative energy possibility.

Psychological correlates of patients with postural orthostatic tachycardia syndrome (POTS).

Psychological symptoms are frequently reported in patients with Postural Orthostatic Tachycardia Syndrome (POTS); however, the nature of these symptoms is not well understood. The current study described baseline psychological symptoms in patients with POTS, and examined associations between psychological and self-report autonomic symptoms. Participants reported mild anxiety symptoms, moderate depressive symptoms, severe somatization, and elevated anxiety sensitivity. Depressive symptoms and pain catastrophizing were significantly associated with autonomic symptoms. The current study adds to the literature by documenting elevated levels of anxiety sensitivity, and relationships between psychological and autonomic symptoms.

Unexpected Short-Tandem-Repeat Patterns in Posttransplant Chimerism Testing: Investigation of 3 Cases with Help from Forensic Science.

Chimerism testing by short tandem repeats (STRs) is used to monitor engraftment after allogeneic hematopoietic stem cell transplantation (HSCT). Generally, STR alleles are stable and transferred from parent to child or from donor to recipient. However, 3 cases did not follow this norm. Additional work-up with help from forensic literature solved these mysteries. In case 1, the patient received HSCT from his son. The son shared STR alleles in 22/23 loci except Penta E, which was explained by repeat expansion in the son. In case 2, the patient had been in remission for 14 years after HSCT for lymphoma and developed repeat expansion in CSF1PO in granulocytes. In case 3, a pre-HSCT patient demonstrated 3 alleles, with 2 peaks taller than the third, in the FGA locus (chromosome 4). A combination of a triallelic variant and leukemia-associated trisomy 4 explained the finding. STR number variants are rare and clinically inconsequential but can overlap malignancy-associated, clinically significant changes.

A novel null allele, HLA-DPA1*01:35N in a European American Male from Pennsylvania, USA.

A non-sense mutation in either DPA1*01:03:01:02 or DPA1*01:03:01:05/01:03:01:15 results in the novel allele, HLA-DPA1*01:35N.

Inter-clinician eConsults without programmatic incentives or requirements: a qualitative study of primary care provider perspectives.

BACKGROUND: Inter-clinician electronic consultation (eConsult) programmes are becoming more widespread in the USA as health care systems seek innovative ways of improving specialty access. Existing studies examine models with programmatic incentives or requirements for primary care providers (PCPs) to participate. OBJECTIVE: We aimed to examine PCP perspectives on eConsults in a system with no programmatic incentive or requirement for PCPs to use eConsults. METHODS: We conducted seven focus groups with 41 PCPs at a safety-net community teaching health care system in Eastern Massachusetts, USA. RESULTS: Focus groups revealed that eConsults improved PCP experience by enabling patient-centred care and enhanced PCP education. However, increased workload and variations in communication patterns added challenges for PCPs. Patients were perceived as receiving timelier and more convenient care. Timelier care combined with direct documentation in the patient record was perceived as improving patient safety. Although cost implications were less clear, PCPs perceived costs as being lowered through fewer unnecessary visits and laboratories. CONCLUSIONS: Our findings suggest that eConsult systems with no programmatic incentives or requirements for PCPs have the potential to improve care.

Incorporating patient input into the design of a disease management program for COPD.

In the era of value-based payment contracts, there is increasing emphasis on disease management as a strategy for improving care quality and reducing costs. To design effective disease management programs, healthcare systems should understand the day-to-day experience of living with particular health conditions, and ensure that evidence-based services and interventions are adapted to align with the realities of patients' lives and their priorities. For healthcare systems operating with limited resources, there is a need for practical and small-scale approaches for collecting and using patient input as part of program design and operations. This case study describes a targeted interview process that Cambridge Health Alliance (CHA) used to gather patient input during the design of a disease management program for chronic obstructive pulmonary disease. The patient perspectives gathered through the interviews influenced several aspects of the program design. The key lessons from CHA's experience are: 1) A small-scale approach with cycles of 5-10 interviews can produce valuable insights for program design; 2) Short patient vignettes can be used to summarize patient data in a simple and compelling format; and 3) Clinicians' perspectives are critical for interpreting patient input and extracting information that is most likely to be useful for program design. CHA's approach provides an example of a systematic and practical process for gathering patient input that other healthcare systems can adapt to their local contexts.

Chemical Profiling of Volatile Organic Compounds in the Headspace of Algal Cultures as Early Biomarkers of Algal Pond Crashes.

Algae ponds used in industrial biomass production are susceptible to pathogen or grazer infestation, resulting in pond crashes with high economic costs. Current methods to monitor and mitigate unhealthy ponds are hindered by a lack of early indicators that precede culture crash. We used solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS) to identify volatiles emitted from healthy and rotifer infested cultures of Microchloropsis salina. After 48 hours of algal growth, marine rotifers, Brachionus plicatilis, were added to the algae cultures and volatile organic compounds (VOC) were sampled from the headspace using SPME fibers. A GC-MS approach was used in an untargeted analysis of VOCs, followed by preliminary identification. The addition of B. plicatilis to healthy cultures of M. salina resulted in decreased algal cell numbers, relative to uninfected controls, and generated trans-ß-ionone and ß-cyclocitral, which were attributed to carotenoid degradation. The abundances of the carotenoid-derived VOCs increased with rotifer consumption of algae. Our results indicate that specific VOCs released by infected algae cultures may be early indicators for impending pond crashes, providing a useful tool to monitor algal biomass production and pond crash prevention.

Personalized Chimerism Test that Uses Selection of Short Tandem Repeat or Quantitative PCR Depending on Patient's Chimerism Status.

Chimerism testing is used to monitor engraftment and risk of relapse after allogeneic hematopoietic stem cell transplantation for hematologic malignancies. Although short tandem repeat (STR) method is widely used among clinical laboratories, quantitative PCR (qPCR) provides better sensitivity (0.1%) than STR (1% to 5%) but is less accurate than STR for patients in mixed chimerism. qPCR chimerism allows evaluation of residual recipient cells as a surrogate of measurable residual disease. To achieve higher sensitivity and accuracy, we applied qPCR or STR based on patient chimerism status (recipient alleles <5% or ≥5%, respectively). Of the 230 patients tested by STR in a 1-year period, excluding 10 deceased patients, 30 qPCR markers were genotyped and 167 patients converted to qPCR chimerism (76%), including eight patients undergoing multiple-donor transplantation. STR was continued on 53 patients (24%) for the following reasons: mixed chimerism (n = 23), lack of donor or pretransplantation DNA (n = 22), and insufficient qPCR informative markers [8 of 60 patients with related donors (13.3%)]. qPCR detected residual recipient chimerism in 85.5% of patients with complete chimerism by STR (<5% recipient). Selecting STR or qPCR testing based on each patient's chimerism status facilitates sensitive and accurate chimerism testing in clinical settings. In addition, we discuss clinical relevance of chimerism testing for measurable residual disease detection in various hematologic malignancies.

CEO-CLER Innovation Grants Program: Empowering Residents as Clinical Learning Environment Change Agents.

BACKGROUND: Many efforts over the past decade have focused on developing quality improvement and safety curricula for residents. Sponsoring institutions have encountered challenges aligning resident projects with institutional quality and safety priorities, engaging faculty mentors, and securing support for resident initiatives from executive leadership. OBJECTIVE: We developed a small grants program to support resident-led change projects intended to improve the clinical learning environment. We assessed program acceptability to residents and faculty, impact of program structure in supporting successful change projects, and program feasibility and financial sustainability. METHODS: Program acceptability was assessed through a review of resident participation. Three aspects of resident change project success were considered: (1) accomplishment of stated aims; (2) institutional change beyond the end of grant funding; and (3) academic publication or presentation. The impact of program structure on project success was assessed through a review of submitted end-of-year narrative reports. RESULTS: The Award Selection Committee has given 41 awards to 44 residents over 4 years, engaging 21% (44 of 213) of residents. Seventy-one percent of projects (29 of 41) produced changes that continued beyond the grant year, and 46% (19 of 41) produced an academic publication or presentation. At the end of the grant period that funded the program's initial 3 years, the chief executive officer elected to continue program funding. CONCLUSIONS: A small grants program supporting resident-led change projects intended to improve the clinical learning environment is acceptable to residents and faculty, feasible to administer, and sustainable with support from institutional senior leaders.

Advances in multiplex nucleic acid diagnostics for blood-borne pathogens: promises and pitfalls - an update.

INTRODUCTION: Multiplex nucleic acid diagnostics for blood-borne pathogens have moved closer to clinical application in the two years since we first reviewed this topic. Areas covered: A new emphasis on detecting pathogens directly in a blood sample without culture, coupling PCR amplification to microfluidic devices and higher multiplexing in isothermal amplification are some of the advances. A wholly new approach of correlating host gene expression response with specific infectious agents opens another opportunity for multiplex detection. Established microarrays, which had been the highest multiplicity platform, are being displaced by Next Generation Sequencing (NGS) having potentially no limit to the number of pathogens that it can identify. Greater accessibility of sequencing devices, standardization of bioinformatic analysis pathways and increased acceptance from regulatory authorities are driving this technology. Expert commentary: The landscape of traditional diagnostics for detection of blood-borne pathogens has changed in the last 5 years. There is no doubt that NSG is recognized as a disruptive technology with a growing repertoire of tools, such as subtyping, resistome analysis, etc., available for clinical microbiology. Increasing acceptance indicates the dominating position of NGS as the future of multiplex molecular diagnostics for blood-borne pathogens.