Fast and accurate diagnosis of bloodstream infections (BSIs) is critical for administering an appropriate treatment regimen in patients, who face hourly-increasing mortality risk due to sepsis. The current gold standard technique is blood culture, and it takes 15 to 72 hours for pathogen identification (ID) and an additional 8 to 15 hours for antimicrobial susceptibility (AST). Apart from the long turnaround time, blood culture is prone to false positives and negatives. Most of the nucleic acid-based methods in the market also require blood culture as a starting sample and/or are limited in identifying pre-defined set of pathogens and antimicrobial resistance (AMR) genes. Hence, the availability of a rapid, agnostic pathogen diagnostic for sepsis which detects pathogens and associated AMR directly from whole blood in a clinically actionable turnaround time, remains a major unmet need in managing critically ill patients. Towards advancing the field of sepsis diagnostics, we have developed a cutting-edge approach called ASPIRE: Agnostic Sepsis Pathogen Identification and REsistance determination. This workflow is rapid, agnostic, quantitative, automation amenable and based on nanopore sequencing that can report pathogen ID and AMR determinants, directly from whole blood, within 6 to 8 hours. The workflow is illustrated in figure 1. We evaluated the performance of ASPIRE workflow using 3 to 5 mL contrived blood samples (n=50) spiked with typical BSI bacteria and fungi at 10 to 100 CFU/mL. We also tested =1.0 mL clinical samples (n=50 confirmed positives and 10 confirmed negatives, tested as blinded samples) to evaluate the clinical concordance of the ASPIRE workflow with blood culture outcomes. The ASPIRE workflow can detect 10-100 CFU/mL of spiked bacteria and fungi in 6 to 8 hours turnaround time. We have achieved removal of 95 to 99% human genomic DNA from 3 mL blood samples. Multiple AMR determinants were also detected within blinded clinical samples. We observed significant concordance with clinical samples. ASPIRE is a novel, cutting-edge, rapid, and universal Sepsis diagnostic workflow that can identify pathogens and characterize their AMR, directly from whole blood. Clinical studies showed significant concordance between blood cultures and ASPIRE workflows. This work has been supported by NIH-NIAID funding under the contract 75N93023C00025

Diagnostics are widely considered crucial in the fight against antimicrobial resistance (AMR), which is expected to kill 10 million people annually by 2030. Nevertheless, there remains a substantial gap between the need for AMR diagnostics versus their development and implementation. To help address this problem, target product profiles (TPP) have been developed to focus developers’ attention on the key aspects of AMR diagnostic tests. However, during discussion between a multisectoral working group of 51 international experts from industry, academia and healthcare, it was noted that specific AMR-related TPPs could be extended by incorporating the interdependencies between the key characteristics associated with the development of such TPPs. Subsequently, the working group identified 46 characteristics associated with six main categories (ie, Intended Use, Diagnostic Question, Test Description, Assay Protocol, Performance and Commercial). The interdependencies of these characteristics were then identified and mapped against each other to generate new insights for use by stakeholders. Specifically, it may not be possible for diagnostics developers to achieve all of the recommendations in every category of a TPP and this publication indicates how prioritising specific TPP characteristics during diagnostics development may influence (or not) a range of other TPP characteristics associated with the diagnostic. The use of such guidance, in conjunction with specific TPPs, could lead to more efficient AMR diagnostics development.

Graphene Field-Effect Transistors In article 2201945, Matthew B. Coppock, Brett Goldsmith, Kiana Aran, and co-workers develop a single multiomics test for detection of respiratory diseases using scalable graphene-based transistors. The embrace of complex, real-time multiomics data, available via graphene-based transistors - converted into human understandable information with the aid of bioinformatics. This can be conceived of as the internet of biology.

The SARS‐CoV‐2 pandemic caused a public health crisis throughout the world and highlighted the need for rapid and sensitive testing as a countermeasure. A sensitive and specific biosensor platform is developed for the detection of antigen and RNA of SARS‐CoV‐2, and its variant (B1.1.529). The demonstrated biosensor platform combines unique protein catalyzed capture bioreceptors (PCCs) for antigen capture and a chimeric (RNA‐DNA) probe for RNA detection using LwaCas13a collateral cleavage activity atop graphene field effect transistors (gFETs). The reported biosensor is able to differentiate unprocessed 10 4 pfu m −1 samples of SARS‐CoV‐2 from Influenza and Rhinovirus. The limit of detection (LOD) calculated for SARS‐CoV‐2 antigen is 10 3 in buffer and 10 4 PFU mL −1 in 10% saliva, while LOD of ≈65 a m calculated for viral RNA isolate without amplification. To provide a high reliability of detection, the role of internal and external factors with respect to gate voltage is further analyzed by Principal Component Analysis (PCA). Based on PCA analysis, the authors are able to classify the samples as pathogen positive or negative ( Y > 0: Positive for pathogen, Y < 0: Negative for pathogen). The reported platform can be quickly adapted for multi‐omics and multiplexed diagnosis of continuously evolving biothreats and global pandemics.

Most people around the world do not have access to facility-based diagnostic testing and the gap in availability of diagnostic tests is a major public health challenge. Testing outside conventional clinical settings are transforming infectious disease diagnostic testing especially in low- and middle-income countries (LMICs). We conducted a systematic review and meta-analysis, searching six databases and including original research manuscripts comparing testing outside clinics with conventional testing. Main outcomes were test uptake and linkage to care, delivery models and adverse outcomes. We identified 76 studies which were included. Data from 18 studies were pooled in meta-analyses. Studies focused on HIV (48), chlamydia (8), and multiple diseases (20). HIV self-testing increased test uptake compared with facility-based testing (nine studies, pooled OR 2.59, 95%CI = 1·06–6·29, moderate quality). STI self-sampling increased test uptake compared with facility-based testing (seven studies, pooled OR 1.74 95% CI=0.97 – 3.12, moderate quality). Innovative and other digital test delivery services improved test uptake compared with facility-based testing services. We defined digital as emails, websites, instant messaging, or related internet approaches. Our work shows testing outside of clinics increased test uptake without significant adverse outcomes. These testing approaches provide an opportunity to expand access and empower patients through innovative delivery models. From the policy viewpoint, this work demonstrates that the use of digital technology in testing outside clinics is a useful strategy for improving infectious diseases screening and linkage to care, and policies that aim to promote the use of these strategies are needed. These models may be especially useful for diseases associated with stigmatization such as HIV and other STIs in LMICs. However, inherent challenges include ensuring linkage to care continuum, quality assured testing and getting the right tests to the right people. Further implementation research and scale-up of effective decentralized models in LMIC settings is needed.

Background: HIV self-testing (HIVST) is recommended by the WHO as an innovative strategy to reach UNAIDS targets to end HIV by 2030. HIVST with digital supports is defined as the use of digital interventions (e.g., website-based, social media, mobile HIVST applications (apps), text messaging (SMS), digital vending machines (digital VMs)) to improve the efficiency and impact of HIVST. HIVST deployment and integration in health services is an emerging priority. We conducted a systematic review aiming to close the gap in evidence that summarizes the impact of digitally supported HIVST and to inform policy recommendations. Methods: We searched PubMed and Embase for articles and abstracts on HIVST with digital supports published during the period February 1st, 2010 to June 15th, 2021, following Cochrane guidelines and PRISMA methodology. We assessed feasibility, acceptability, preference, and impact outcomes across all populations and study designs. Metrics reported were willingness to use HIVST, preferences for HIVST delivery, proportion of first-time testers, HIVST uptake, HIVST kit return rate, and linkage to care. Heterogeneity of the interventions and reported metrics precluded us from conducting a meta-analysis. Findings: 46 studies were narratively synthesized, of which 72% were observational and 28% were RCTs. Half of all studies (54%, 25/46) assessed web-based innovations (e.g., study websites, videos, chatbots), followed by social media (26%, 12/46), HIVST-specific apps (7%, 3/46), SMS (9%, 4/46), and digital VMs (4%, 2/46). Web based innovations were found to be acceptable (77-97%), preferred over in-person and hybrid options by more first-time testers (47-48%), highly feasible (93-95%), and were overall effective in supporting linkage to care (53-100%). Social media and app-based innovations also had high acceptability (87-95%) and linkage to care proportions (80-100%). SMS innovations increased kit return rates (54-94%) and HIVST uptake among hard-to-reach groups. Finally, digital VMs were highly acceptable (54-93%), and HIVST uptake was six times greater when using digital VMs compared to distribution by community workers. Interpretation: HIVST with digital supports was deemed feasible, acceptable, preferable, and was shown to increase uptake, engage first-time testers and hard-to-reach populations, and successfully link participants to treatment. Findings pave the way for greater use of HIVST interventions with digital supports globally. Funding: This work was funded by the Foundation for Innovative New Diagnostics. The agency had no role in the decision to submit it for publication, however the funders contributing to the writing of the manuscript. NPP also acknowledges support from the Fonds de recherche du Quebec Sante (Senior scientist scholar award), The Canadian Institutes of Health Research (PJT 153149 and HBR 422155), Grand Challenges Canada (Transition to Scale award, 071005), the India-Canada centre for Innovative Multidisciplinary Partnerships to Accelerate Community Transformation and Sustainability (IC-IMPACTS), and the MUHC Foundation. (c) 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

With many regions of the world seeing a rise in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases, borders, schools, and workplaces are partially or fully closed, and physical interactions are restricted. Although vaccine roll-out has begun in numerous countries,1British Broadcasting CompanyCovid-19 vaccine: first person receives Pfizer jab in UK.https://www.bbc.co.uk/news/uk-55227325Date accessed: December 15, 2020Google Scholar it will take many months to complete. Meanwhile, communities are reliant on diagnostic testing in conjunction with other public health measures to keep facilities open. Until now, testing for SARS-CoV-2 has been mostly limited to use for clinical confirmation and care, but some countries are now implementing widespread testing for public health use and risk management. However, there is little guidance available to policy makers on how to translate the scientific information on SARS-CoV-2 diagnostic tests into practical policies for widespread testing in non-clinical settings. The development of rapid antigen tests, which can be used outside of the laboratory with fast turnaround times, has made widespread testing feasible. Although there were initial concerns about test performance, emerging evidence suggests that the most sensitive tests can detect 97% of infectious cases, based on the relationship between viral load and infectiousness.2van Beek J Igloi Z Boelsums T et al.From more testing to smart testing: data-guided SARS-CoV-2 testing choices.medRxiv. 2020; (published online Oct 14.) (preprint)https://doi.org/10.1101/2020.10.13.20211524Google Scholar Moreover, modelling analyses show that frequency of testing and speed of reporting have a greater influence than test sensitivity on the effectiveness of SARS-CoV-2 infection surveillance.3Larremore DB Wilder B Lester E et al.Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance.medRxiv. 2020; (published online June 27.) (preprint)https://doi.org/10.1101/2020.06.22.20136309PubMed Google Scholar Although the nasopharyngeal specimens required for many antigen tests are challenging to obtain, tests that utilise alternative sampling methods, such as nasal or saliva specimens, are becoming available and could provide the ease of use necessary for widespread testing.4Lindner AK Nikolai O Kausch F et al.Head-to-head comparison of SARS-CoV-2 antigen-detecting rapid test with self-collected anterior nasal swab versus professional-collected nasopharyngeal swab.Eur Respir J. 2020; (published online Dec 10.)https://doi.org/10.1183/13993003.03961-2020Crossref PubMed Scopus (104) Google Scholar, 5Azzi L Baj A Alberio T et al.Rapid salivary test suitable for a mass screening program to detect SARS-CoV-2: a diagnostic accuracy study.J Infect. 2020; 81: e75-e78Summary Full Text Full Text PDF PubMed Scopus (51) Google Scholar These core principles (ie, the importance of fast test turnaround times, the relationship between test positivity and infectivity, and the ease of use for sample colletion) are applicable to all SARS-CoV-2 testing policies for public health use, along with cost considerations relating to high-volume testing. Beyond the core principles, several factors specific to the setting in which the test is to be used should be considered when designing testing policies. Settings can be defined according to patterns of contact and level of exposure, and include: points of entry, where people transit across defined borders; semi-closed communities, where the same people come together repeatedly; and random exposure settings, where people meet for a single exposure and then disperse (figure). The testing intensity required for entry points differs according to the level of risk of spread, which varies according to the frequency and distance of travel, and the prevalence of disease. The mode of travel strongly influences its frequency; air travel is usually planned and less frequent, whereas land crossings are more spontaneous and regular, even occurring daily. Prevalence of disease has a greater impact on risk when individuals cross from a higher prevalence area into a lower prevalence area, compared with similar prevalence areas. Long distance journeys have additional risk associated with the period of travel, as they are likely to entail means of transport involving many people (eg, aeroplanes, buses, or trains). Additionally, quarantine restrictions might need to be more flexible for commercial vehicles than for non-commercial vehicles, for economic reasons. Currently, strategies for reducing transmission in semi-closed communities are reliant on public health measures, such as physical distancing and mask wearing, supplemented by testing symptomatic individuals and contacts in health-care settings in line with country policies. There is still a potential for outbreaks in these communities, leading to quarantines or temporary closures. Strategies for additional proactive testing to detect asymptomatic infections and prevent spread can range from testing of individuals who are at high risk of being exposed to the virus (eg, those in frontline roles), to mass-testing the whole community. Testing in semi-closed communities is more effective at preventing outbreaks when done frequently, especially for individuals with a high risk of exposure,3Larremore DB Wilder B Lester E et al.Test sensitivity is secondary to frequency and turnaround time for COVID-19 surveillance.medRxiv. 2020; (published online June 27.) (preprint)https://doi.org/10.1101/2020.06.22.20136309PubMed Google Scholar, 6Park SY Kim YM Yi S et al.Coronavirus disease outbreak in call center, South Korea.Emerg Infect Dis. 2020; 26: 1666-1670Crossref PubMed Scopus (339) Google Scholar but the optimal frequency of testing is dependent on community-specific factors, such as total population, proportion of the population who consistently wear masks, and presence of a contact tracing programme. Random exposure settings are particularly susceptible to the cluster-based superspreader events characteristic of COVID-19. Risk can be reduced by preventing the virus from entering the three Cs: crowded places, close contact settings, and confined and enclosed spaces.7WHOCoronavirus disease (COVID-19) advice for the public.https://www.who.int/emergencies/diseases/novel-coronavirus-2019/advice-for-publicDate accessed: November 30, 2020Google Scholar However, risk limitation becomes more challenging as the size of a gathering increases, because physical distancing becomes more difficult in a large crowd and individuals are likely to have travelled from areas with a higher prevalence of infection.8Centers for Disease Control and PreventionConsiderations for events and gatherings.https://www.cdc.gov/coronavirus/2019-ncov/community/large-events/considerations-for-events-gatherings.htmlDate accessed: November 30, 2020Google Scholar Practically, reducing amplification events in these settings would need participants to be tested before arrival, which would require diagnostic tests to be readily available to the public outside of usual health-care settings. Although communities are progressing towards decentralised testing,9United States Food and Drug AdministrationLucira COVID-19 All-In-One Test Kit.https://www.fda.gov/media/143810/downloadDate accessed: November 26, 2020Google Scholar current tools are not sufficiently reliable or affordable for routine and repeated use. Additionally, a test for use outside of a health-care setting would need to incorporate a method of presenting results, with certification to ensure authenticity (especially in light of an already emerging market for fake SARS-CoV-2 test certificates10The ConnexionGroup caught making fake Covid test results at Paris airport.https://www.connexionfrance.com/French-news/Group-caught-making-fake-Covid-test-results-at-Paris-airportDate accessed: December 8, 2020Google Scholar). Digital integration would be crucial to such a strategy. Ultimately, commoditised and reliable home-based testing available at low cost would be key to allowing population-level mass testing. Robust strategies for widespread testing should take into account the core principles relating to diagnostic testing, and specific considerations for each defined use. Evidence suggests that rapid antigen tests are relevant for a wide set of uses, but there is still a need to invest in the development of novel diagnostics for commoditised use beyond the health-care system. CB convenes to and EH manages the ACT-Accelerator Diagnostics Pillar on behalf of FIND and the Global Fund. RS declares no competing interests. We thank Rachel Wright, PhD, Rachel Wright Medical Communications (Crewe, UK)for providing medical writing services, funded by Foundation for Innovative New Diagnostics (FIND), in accordance with Good Publication Practice (GPP3).

Background. Most people around the world do not have access to facility-based diagnostic testing, and the gap in availability of diagnostic tests is a major public health challenge. Self-testing, self-sampling, and institutional testing outside conventional clinical settings are transforming infectious disease diagnostic testing in a wide range of low- and middle-income countries (LMICs). We examined the delivery models of infectious disease diagnostic testing outside clinics to assess the impact on test uptake and linkage to care. Methods. We conducted a systematic review and meta-analysis, searching 6 databases and including original research manuscripts comparing testing outside clinics with conventional testing. The main outcomes were test uptake and linkage to care, delivery models, and adverse outcomes. Data from studies with similar interventions and outcomes within thematic areas of interest were pooled, and the quality of evidence was assessed using GRADE. This study was registered in PROSPERO (CRD42019140828). We identified 10 386 de-duplicated citations, and 76 studies were included. Data from 18 studies were pooled in meta-analyses. Studies focused on HIV (48 studies), chlamydia (8 studies), and multiple diseases (20 studies). HIV self-testing increased test uptake compared with facility-based testing (9 studies: pooled odds ratio [OR], 2.59; 95% CI, 1.06-6.29; moderate quality). Self-sampling for sexually transmitted infections increased test uptake compared with facility-based testing (7 studies: pooled OR, 1.74; 95% CI, 0.97-3.12; moderate quality). Conclusions. Testing outside of clinics increased test uptake without significant adverse outcomes. These testing approaches provide an opportunity to expand access and empower patients. Further implementation research, scale-up of effective service delivery models, and policies in LMIC settings are needed.

ObjectivesBlood culture results inadequately stratify the mortality risk in critically ill patients with sepsis. We sought to establish the prognostic significance of the presence of microbial DNA in the bloodstream of patients hospitalized with suspected sepsis.MethodsWe analysed the data collected during the Rapid Diagnosis of Infections in the Critically Ill (RADICAL) study, which compared a novel culture-independent PCR/electrospray ionization-mass spectrometry (ESI-MS) assay with standard microbiological testing. Patients were eligible for the study if they had suspected sepsis and were either hospitalized or were referred to one of nine intensive care units from six European countries. The blood specimen for PCR/ESI-MS assay was taken along with initial blood culture taken for clinical indications.ResultsOf the 616 patients recruited to the RADICAL study, 439 patients had data on outcome, results of the blood culture and PCR/ESI-MS assay available for analysis. Positive blood culture and PCR/ESI-MSI result was found in 13% (56/439) and 40% (177/439) of patients, respectively. Either a positive blood culture (p 0.01) or a positive PCR/ESI-MS (p 0.005) was associated with higher SOFA scores on enrolment to the study. There was no difference in 28-day mortality observed in patients who had either positive or negative blood cultures (35% versus 32%, p 0.74). However, in patients with a positive PCR/ESI-MS assay, mortality was significantly higher in comparison to those with a negative result (42% versus 26%, p 0.001).ConclusionsPresence of microbial DNA in patients with suspected sepsis might define a patient group at higher risk of death.

Initial antimicrobial treatment of patients with deep seated or invasive infections is typically empiric. Usually, cultures of specimens obtained from the suspected source of infection are performed to identify pathogens and guide continued antimicrobial treatment. When patients present with signs and symptoms of infection, but sterile body fluid or tissue specimens cannot be obtained in a timely fashion, growth of bacterial pathogens in culture may be inhibited following initiation of empiric antibiotic treatment. To address this clinical dilemma, we performed a prospective evaluation of conventional culture vs. PCR coupled to electrospray ionization mass spectrometry (PCR/ESI-MS) on sterile body fluids and tissues submitted to the diagnostic microbiology lab following initiation of empiric antibiotic treatment for patients with suspected infection. In this series of surgical samples, PCR/ESI-MS identified bacterial pathogen(s) in 56% (49/87) of patients with non-diagnostic cultures. Examination of patients stratified by antibiotic treatment duration demonstrated that PCR/ESI-MS sustains high rates of bacterial DNA detection over time by generalized estimating equation models (p<0.0001).

Identifying the pathogen responsible for culture-negative valve endocarditis often depends on molecular studies performed on surgical specimens. A patient with Ehlers-Danlos syndrome who had an aortic graft, a mechanical aortic valve, and a mitral anulloplasty ring presented with culture-negative prosthetic valve endocarditis and aortic graft infection. Research-based polymerase chain reaction (PCR)/electrospray ionization mass spectrometry on peripheral blood samples identified Bartonella henselae. Quantitative PCR targeting the16S-23S ribonucleic acid intergenic region and Western immunoblotting confirmed this result. This, in turn, permitted early initiation of pathogen-directed therapy and subsequent successful medical management of B henselae prosthetic valve endocarditis and aortic graft infection.

Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States.

e314 www.ccmjournal.org May 2016 • Volume 44 • Number 5 The authors reply: We thank Moorman et al (1) for their interest in our study (2) and their appreciation for the necessity of advance in evaluation of organ system network. We agree multidisciplinary collaborations with more sophisticated mathematical analysis will further develop our understanding for the complexity and dynamics of each organ system function in sepsis and that time-series evaluation of the organ system network is necessary for improvement of our clinical management of sepsis. As Moorman et al (1) pointed out, our study (2) demonstrated more disrupted organ system network in nonsurvivors just at the moment of ICU admission with the specific combination of representative variables. Certainly, a new analytical strategy incorporated with network physiology (3), which focuses on dynamical aspects of organ system network, will enable to identify disruption of network in sepsis with more sensitivity. We agree that real-time continuous monitoring can predict exacerbation of septic state before clinical symptoms appear and may tell us the optimal timing of certain therapeutic interventions. Godin and Buchman (4) indicated that inappropriate and excessive secretion of cytokines and inflammatory mediators, recognized as systemic inflammatory response syndrome, might provoke organ system network deterioration. In addition to sepsis, these changes in humeral mediators accompany with other critical conditions such as surgery, trauma, burn, and pancreatitis (5, 6). Even commonly performed medical interventions including mechanical organ support and blood transfusion may cause inflammatory response (7, 8). Thus, continuous monitoring of networked organ system interactions will have great merits for evaluation of critically ill patients. Further investigation incorporating multidisciplinary approach by intensive care medicine and network physiology is expected to provide more clear view of clinical course of illness and contribute to develop novel diagnostics and therapeutics in the fight against critical illnesses including sepsis. The authors have disclosed that they do not have any potential conflicts of interest.

Copyright © by 2016 by the Society of Critical Care Medicine and Wolters Kluwer Health, Inc. All Rights Reserved.

BACKGROUND:Rapid molecular diagnostic (RMD) platforms may lead to better antibiotic use. Our objective was to develop analytical strategies to enhance the interpretation of RMDs for clinicians. METHODS:We compared the performance characteristics of 4 RMD platforms for detecting resistance against β-lactams in 72 highly resistant isolates of Escherichia coli and Klebsiella pneumoniae (PRIMERS I). Subsequently, 2 platforms were used in a blinded study in which a heterogeneous collection of 196 isolates of E. coli and K. pneumoniae (PRIMERS II) were examined. We evaluated the genotypic results as predictors of resistance or susceptibility against β-lactam antibiotics. We designed analytical strategies and graphical representations of platform performance, including discrimination summary plots and susceptibility and resistance predictive values, that are readily interpretable by practitioners to inform decision-making. RESULTS:In PRIMERS I, the 4 RMD platforms detected β-lactamase (bla) genes and identified susceptibility or resistance in >95% of cases. In PRIMERS II, the 2 platforms identified susceptibility against extended-spectrum cephalosporins and carbapenems in >90% of cases; however, against piperacillin/tazobactam, susceptibility was identified in <80% of cases. Applying the analytical strategies to a population with 15% prevalence of ceftazidime-resistance and 5% imipenem-resistance, RMD platforms predicted susceptibility in >95% of cases, while prediction of resistance was 69%-73% for ceftazidime and 41%-50% for imipenem. CONCLUSIONS:RMD platforms can help inform empiric β-lactam therapy in cases where bla genes are not detected and the prevalence of resistance is known. Our analysis is a first step in bridging the gap between RMDs and empiric treatment decisions.

Objective: Early identification of causative microorganism(s) in patients with severe infection is crucial to optimize antimicrobial use and patient survival. However, current culture-based pathogen identification is slow and unreliable such that broad-spectrum antibiotics are often used to insure coverage of all potential organisms, carrying risks of overtreatment, toxicity, and selection of multidrug-resistant bacteria. We compared the results obtained using a novel, culture-independent polymerase chain reaction/electrospray ionization-mass spectrometry technology with those obtained by standard microbiological testing and evaluated the potential clinical implications of this technique. Design: Observational study. Setting: Nine ICUs in six European countries. Patients: Patients admitted between October 2013 and June 2014 with suspected or proven bloodstream infection, pneumonia, or sterile fluid and tissue infection were considered for inclusion. Interventions: None. Measurements and Main Results: We tested 616 bloodstream infection, 185 pneumonia, and 110 sterile fluid and tissue specimens from 529 patients. From the 616 bloodstream infection samples, polymerase chain reaction/electrospray ionization-mass spectrometry identified a pathogen in 228 cases (37%) and culture in just 68 (11%). Culture was positive and polymerase chain reaction/electrospray ionization-mass spectrometry negative in 13 cases, and both were negative in 384 cases, giving polymerase chain reaction/electrospray ionization-mass spectrometry a sensitivity of 81%, specificity of 69%, and negative predictive value of 97% at 6 hours from sample acquisition. The distribution of organisms was similar with both techniques. Similar observations were made for pneumonia and sterile fluid and tissue specimens. Independent clinical analysis of results suggested that polymerase chain reaction/electrospray ionization-mass spectrometry technology could potentially have resulted in altered treatment in up to 57% of patients. Conclusions: Polymerase chain reaction/electrospray ionization-mass spectrometry provides rapid pathogen identification in critically ill patients. The ability to rule out infection within 6 hours has potential clinical and economic benefits.

The cornerstone of sepsis management requires identifying the causative pathogen and initiating appropriate antimicrobial therapy. Current pathogen detection relies on culture techniques, a technology that is over 100 years old, slow and unreliably. It is not unusual for only 10% of critical care blood cultures to be positive. Due to the low yield and time taken to obtain a result, they rarely alter patient management. a novel molecular pathogen detection system, known as IRIDICA, employs polymerase chain reaction and electro spray ionisation mass spectroscopy (PCR/ESI-MS) to identify over 1000 pathogens, direct from sample without culture and within 8 hours. The RADICAL study was created to assess this technology in a real world critical care environment.

Broad-range 16S ribosomal RNA gene PCR coupled with Sanger sequencing was originally employed by soil scientists and was subsequently adapted for clinical applications. PCR coupled with electrospray ionization mass spectrometry has also progressed from initial applications in the detection of organisms from environmental samples into the clinical realm and has demonstrated promise in detection of pathogens in clinical specimens obtained from patients with suspected infection but negative cultures. We review studies of multiplex PCR, 16S ribosomal RNA gene PCR and sequencing and PCR coupled with electrospray ionization mass spectrometry for detection of bacteria in specimens that were obtained from patients during or after administration of antibiotic treatment, and examine the role of each for assisting in antimicrobial treatment and stewardship efforts. Following an exploration of the available data in this field, we discuss the opportunities that the preliminary investigations reveal, as well as the challenges faced with the implementation of these strategies in clinical practice.

ABSTRACT The widespread dissemination of carbapenem-resistant Acinetobacter spp. has created significant therapeutic challenges. At present, rapid molecular diagnostics (RMDs) that can identify this phenotype are not commercially available. Two RMD platforms, PCR combined with electrospray ionization mass spectrometry (PCR/ESI-MS) and molecular beacons (MB), for detecting genes conferring resistance/susceptibility to carbapenems in Acinetobacter spp. were evaluated. An archived collection of 200 clinical Acinetobacter sp. isolates was tested. Predictive values for susceptibility and resistance were estimated as a function of susceptibility prevalence and were based on the absence or presence of beta-lactamase (bla) NDM, VIM, IMP, KPC, and OXA carbapenemase genes (e.g., blaOXA-23, blaOXA-24/40, and blaOXA-58 found in this study) against the reference standard of MIC determinations. According to the interpretation of MICs, 49% (n = 98) of the isolates were carbapenem resistant (as defined by either resistance or intermediate resistance to imipenem). The susceptibility sensitivities (95% confidence interval [CI]) for imipenem were 82% (74%, 89%) and 92% (85%, 97%) for PCR/ESI-MS and MB, respectively. Resistance sensitivities (95% CI) for imipenem were 95% (88%, 98%) and 88% (80%, 94%) for PCR/ESI-MS and MB, respectively. PRIMERS III establishes that RMDs can discriminate between carbapenem resistance and susceptibility in Acinetobacter spp. In the context of a known prevalence of resistance, SPVs and RPVs can inform clinicians regarding the best choice for empiric antimicrobial therapy against this multidrug-resistant pathogen.

To the Editor: Each year, nonpolio enteroviruses cause 10–15 million infections in the United States (1). Enterovirus D68 (EV-D68) is an uncommon strain of nonpolio enterovirus that emerged in Illinois and Missouri in August 2014 in association with severe respiratory infections in children and spread across the United States (2). On August 23, 2014, the infection control department for Comer’s Children’s Hospital at the University of Chicago initially notified the Centers for Disease Control and Prevention of an increased number of children hospitalized with unusually severe respiratory illness (3). From mid-August to December 4, 2014, there were 1,121 laboratory-confirmed cases of EV-D68 in the United States (2). Almost all EV-D68 infections have occurred in children, many of whom had a history of asthma or wheezing (2). One day before the first report (August 22, 2014), a 26-year-old obese woman with an unremarkable medical history was transferred to the medical intensive care unit at Saint Francis Medical Center, a tertiary care medical center in Peoria, Illinois, USA, with severe acute respiratory distress syndrome (ARDS). The transfer was from a nearby community hospital where she had sought care 4 days earlier for influenza-like symptoms consisting of cough, wheezing, progressive shortness of breath, nausea, and vomiting. In the community hospital emergency department, she mentioned that 2 children at home had similar symptoms and that her mother had recently been hospitalized with an acute respiratory illness. Despite treatment with supplemental oxygen, nebulized albuterol, and intravenous antimicrobial drugs for community-acquired pneumonia, her condition deteriorated, and she was intubated on hospital day 2, after which the antimicrobial drug treatment was changed from intravenous ceftriaxone and azithromycin to intravenous vancomycin and piperacillin/tazobactam. Results of bronchoscopy performed on hospital day 4 were unremarkable, and bacterial cultures of alveolar lavage samples were negative. Her transfer to St. Francis Medical Center was prompted by persistent mechanical ventilation requirements of 100% fraction of inspired oxygen; positive end-inspiratory pressure of 12 mm/H2O consistent with classic ARDS (hypoxemia, indicated by a ratio of arterial oxygen partial pressure to fractional inspired oxygen <200 mm Hg); and bilateral infiltrates on chest radiograph (Figure) without evidence of left heart failure (4). On hospital day 8 (cumulative), a nasopharyngeal swab sample was tested by FilmArray Respiratory Panel multiplex PCR (BioFire Diagnostics, Salt Lake City, UT, USA); results were positive for rhinovirus/enterovirus. That day, intravenous methylprednisolone therapy was initiated. Figure Chest radiograph obtained (with portable machine) of semirecumbent adult patient with enterovirus D68–associated acute respiratory distress syndrome on hospital day 3. During a prolonged hospital stay, the patient required mechanical ventilation for 32 days, underwent a second bronchoscopic evaluation, required a percutaneous tracheostomy (and subsequent decannulation), and underwent endoscopic gastrostomy tube placement (and removal). She was discharged from the hospital after 55 days and ultimately recovered completely. To determine the etiology of the clinical syndrome for the patient reported here, molecular diagnostic testing of respiratory tract clinical specimens was required. Institutional review board approval was obtained for molecular diagnostics and sequencing of the patient’s nasopharyngeal swab specimens and bronchoalveolar lavage (BAL) fluid samples. The FilmArray platform is capable of detecting enteroviral infections caused by EV-D68 but cannot differentiate between rhinoviruses and enteroviruses (5). Confirmation of EV-D68 requires EV-D68–specific PCR (6). A novel, research-based diagnostic modality that is capable of rapid identification of viral pathogens directly from clinical specimens is the combination of PCR and electrospray ionization mass spectrometry (ESI-MS) (7), which was instrumental in early recognition of the novel pandemic strain of influenza A(H1N1) virus that emerged in 2009 (8). For a variety of viral pathogens, PCR/ESI-MS sensitivity is 94% and specificity is 98% (9). In this case, PCR/ESI-MS detected a human enterovirus from the right middle lobe and left lingular segment BAL fluid samples. For the assay, 2 primer pairs were used; both confirmed the presence of human enterovirus, but only 1 matched the signatures for EV-D68. For confirmation, we pursued testing with EV-D68–specific PCR, which was performed by the Special Projects Laboratory of the Washington University Department of Pediatrics. This assay amplifies a segment of the viral protein 1 gene, which enables discrimination of EV-D68 from other enteroviruses and rhinoviruses (K.M. Wylie et al., unpub. data). The nasopharyngeal swab sample and the right middle lobe and lingula BAL fluid specimens were positive for EV-D68. PCR/ESI-MS of BAL fluid followed by EV-D68–specific PCR testing of 1 nasopharyngeal swab and 2 BAL fluid samples confirmed our clinical suspicion of ARDS secondary to EV-D68 in an adult. The patient’s history of contact with sick family members and clinical signs (nonproductive cough, nausea, and vomiting) were suggestive of a viral infection. Lessons learned from the emergence of swine-origin influenza A(H1N1)pdm09 virus and recognition (in the midst of the pandemic) that younger age and obesity were risk factors for severe disease were also suggestive of a viral respiratory infection. We are developing a specific rapid molecular assay for EV-D68, which should help clinicians recognize when EV-D68 is present in the community. During those times, EV-D68 infection should be included in the differential diagnosis of severe respiratory infection. Documentation of EV-D68 infection may help with clinical management for individual patients and minimize unnecessary use of antimicrobial drugs within communities.