Rapid Molecular Diagnostics Research Articles

More targeted use of oseltamivir and in-hospital isolation facilities after implementation of a multifaceted strategy including a rapid molecular diagnostic panel for respiratory viruses in immunocompromised adult patients

BackgroundImmunocompromised adults are more vulnerable to a complicated course of viral respiratory tract infections (RTI). ObjectivesProvide evidence on the effect of implementation of rapid molecular diagnostics for viruses on use of in-hospital isolation facilities, oseltamivir and antibiotic usage, and other clinical outcomes in immunocompromised patients. Study designA before-after study during two consecutive respiratory viral seasons, including immunocompromised adult patients presenting at a tertiary care emergency department with clinical suspicion of RTI. During the first season (2016/2017), respiratory viruses were detected using inhouse real-time PCR. The second season (2017/2018), we implemented a diagnostic flowchart including a rapid molecular test for 15 respiratory viruses (FilmArray®). We assessed the effect of this implementation on need for isolation, antivirals and empirical antibiotics. ResultsWe included 192 immunocompromised adult patients during the first and 378 during the second season. Respiratory viral testing was performed in 135 patients (70%) during the first and 284 (75%) during the second season (p = 0.218) of which 213 (75%) using the rapid test. After implementation, use of in-hospital isolation facilities was reduced (adjusted odds ratio 0.35, 95%CI 0.19-0.64). Furthermore, adequate use of oseltamivir improved, with fewer prescriptions in influenza negative patients (0.15, 95%CI 0.08-0.28) and more in influenza positive patients (11.13, 95%CI 1.75–70.86). No effect was observed on empirical antibiotic use, hospital admissions, length of hospital stay or safety outcomes. ConclusionsImplementation of rapid molecular testing for respiratory viruses in adult immunocompromised patients results in more adequate use of oseltamivir and in-hospital isolation facilities without compromising safety.

Personalized medicine in chronic kidney disease by detection of monogenic mutations.

A large fraction of early-onset chronic kidney disease (CKD) is known to be monogenic in origin. To date, ∼450 monogenic (synonymous with single-gene disorders) genes, if mutated, are known to cause CKD, explaining ∼30% of cases in pediatric cohorts and ∼5-30% in adult cohorts. However, there are likely hundreds of additional monogenic nephropathy genes that may be revealed by whole-exome or -genome sequencing. Although the discovery of novel CKD-causing genes has accelerated, significant challenges in adult populations remain due to broad phenotypic heterogeneity together with variable expressivity, incomplete penetrance or age-related penetrance of these genes. Here we give an overview of the currently known monogenic causes for human CKD. We also describe how next-generation sequencing facilitates rapid molecular genetic diagnostics in individuals with suspected genetic kidney disease. In an era of precision medicine, understanding the utility of genetic testing in individuals with a suspected inherited nephropathy has important diagnostic and prognostic implications. Detection of monogenic causes of CKD permits molecular genetic diagnosis for patients and families and opens avenues for personalized treatment strategies for CKD. As an example, detection of a pathogenic mutation in the gene HNF1B not only allows for the formal diagnosis of CKD, but can also facilitate screening for additional extrarenal manifestations of disease, such as maturity-onset diabetes of youth, subclinical abnormal liver function tests, neonatal cholestasis and pancreatic hypoplasia. It also provides the driving force towards a better understanding of disease pathogenesis, potentially facilitating targeted new therapies for individuals with CKD.

Underestimated pyrazinamide resistance may compromise outcomes of pyrazinamide containing regimens for treatment of drug susceptible and multi-drug-resistant tuberculosis in Tanzania

BackgroundTuberculosis (TB) is the leading cause of death from an infectious disease and the roll-out of rapid molecular diagnostics for rifampin resistance has resulted in a steady rise in the number of patients with multidrug-resistant (MDR)-TB referred for treatment. Pyrazinamide is used in susceptible TB treatment for 6 months when used in combination with rifampin, isoniazid and ethambutol and is an important companion drug in novel MDR-TB trials. This study was undertaken to determine the prevalence of pyrazinamide resistance by either phenotypic or pncA testing among patients admitted to a referral hospital in Tanzania for drug-susceptible and MDR-TB treatment.MethodsSurveillance sputa were sent among subjects beginning TB therapy at the national MDR-TB referral hospital during a 6 month period in 2013–2014. Mycobacterial cultures of pretreatment sputa were performed at the Kilimanjaro Clinical Research Institute (KCRI) in the BACTEC mycobacterial growth indicator tubes (MGIT) 960 system. Speciation of M. tuberculosis complex was confirmed by MTBc assay. Isolates were sub-cultured on to Lowenstein-Jensen (LJ) slants. Phenotypic resistance to pyrazinamide was performed in the MGIT system while a real-time PCR with High Resolution Melt (HRM) technique was used to determine mutation in the pncA gene from the same pure subculture. Sputa were then collected monthly to determine the time to culture negativity. Final treatment outcome was determined.ResultsNinety-one M. tuberculosis isolates from individual patients were available for analysis of which 30 (32.9%) had MDR-TB, the mean (±SD) age was 33 ± 10 years, and the majority 23 (76.7%) were males. Of the 30 MDR-TB patients, 15(50%) had isolates with pyrazinamide resistance by conventional MGIT testing. This proportion expectedly exceeded the number with pyrazinamide resistance in the 61 patients without MDR-TB, 13 (21.3%) (p = 0.008). Six (20%) of MDR-TB patients had a poor outcome including treatment failure. Among patients with treatment failure, 5 (83%) had pyrazinamide resistance compared to only 10 (41.6%) with treatment success (p = 0.08). Two patients died, and both had isolates with pyrazinamide resistance. No other pretreatment characteristic was associated with treatment outcome.ConclusionPyrazinamide susceptibility appears to be important in clinical outcomes for MDR-TB patients, and susceptibility testing appears to be a critical adjunct to TB care. The high proportion of PZA resistance in non-MDR TB cases calls for further local investigation.

Rapid Molecular Tests for Influenza, Respiratory Syncytial Virus, and Other Respiratory Viruses: A Systematic Review of Diagnostic Accuracy and Clinical Impact Studies.

We systematically reviewed available evidence from Embase, Medline, and the Cochrane Library on diagnostic accuracy and clinical impact of commercially available rapid (results <3 hours) molecular diagnostics for respiratory viruses as compared to conventional molecular tests. Quality of included studies was assessed using the Quality Assessment of Diagnostic Accuracy Studies criteria for diagnostic test accuracy (DTA) studies, and the Cochrane Risk of Bias Assessment and Risk of Bias in Nonrandomized Studies of Interventions criteria for randomized and observational impact studies, respectively. Sixty-three DTA reports (56 studies) were meta-analyzed with a pooled sensitivity of 90.9% (95% confidence interval [CI], 88.7%–93.1%) and specificity of 96.1% (95% CI, 94.2%–97.9%) for the detection of either influenza virus (n = 29), respiratory syncytial virus (RSV) (n = 1), influenza virus and RSV (n = 19), or a viral panel including influenza virus and RSV (n = 14). The 15 included impact studies (5 randomized) were very heterogeneous and results were therefore inconclusive. However, we suggest that implementation of rapid diagnostics in hospital care settings should be considered.

Advances in Rapid Molecular Blood Culture Diagnostics: Healthcare Impact, Laboratory Implications, and Multiplex Technologies.

For far too long, the diagnosis of bloodstream infections has relied on time-consuming blood cultures coupled with traditional organism identification and susceptibility testing. Technologies to define the culprit in bloodstream infections have gained sophistication in recent years, notably by application of molecular methods. In this review, we summarize the tests available to clinical laboratories for molecular rapid identification and resistance marker detection in blood culture bottles that have flagged positive. We explore the cost-benefit ratio of such assays, covering aspects that include performance characteristics, effect on patient care, and relevance to antibiotic stewardship initiatives. Rapid blood culture diagnostics represent an advance in the care of patients with bloodstream infections, particularly those infected with resistant organisms. These diagnostics are relatively easy to implement and appear to have a positive cost-benefit balance, particularly when fully incorporated into a hospital's antimicrobial stewardship program.

1757. Using the Desirability of Outcome Ranking for Management of Antimicrobial Therapy (DOOR-MAT) to Assess Antibiotic Therapy Guided by Rapid Molecular Diagnostics (RMD) in Bloodstream Infection (BSI) Caused by Escherichia coli and Klebsiella pneumoniae

BackgroundIn the setting of Escherichia coli and Klebsiella pneumoniae BSI, empiric antibiotic therapy is determined by clinical judgement and Gram stain (GS) of the positive blood culture bottle up to 2 days before antibiotic susceptibility test (AST) results become available. Within hours of GS, RMD can detect E. coli and K. pneumoniae and predict the pattern of susceptibility to β-lactams (BL) of differing spectrum. In this study, our objective was to compare “real-life” empiric therapy for E. coli and K. pneumoniae BSI administered between GS and AST results with simulated RMD-guided therapy.MethodsWe identified a subset of patients hospitalized within VHA between 2006 and 2015 who had a blood culture positive for E. coli or K. pneumoniae, and received empiric BLs between GS and AST Results. We further restricted the cohort to those with observed or implied AST results for 4 representative BLs: cefazolin, ceftriaxone, piperacillin–tazobactam, and imipenem. We extracted BL resistance patterns and, based on previously analyzed RMD performance on 195 E. coli and K. pneumoniae, we simulated RMD results for our cohort by resampling RMD results stratified by resistance pattern at the observed frequencies of resistance patterns in our clinical isolates. We simulated therapy guided by RMDs and compared with the observed empiric BLs using a DOOR-MAT score (Figure 1).ResultsA total of 36,531 BSI cases were identified. Of these, 9,981 E. coli and 4,545 K. pneumoniae met our inclusion criteria (Figure 2). Among these, susceptibility to all BLs occurred in 88% of cases; resistance to all BLs occurred in <0.5%. The isolates previously analyzed using RMD included more resistant phenotypes (Figure 3). Empiric BLs were active in 98% of BSIs, with a mean DOOR-MAT score of 66.1 and 59% of cases classified as “moderate overtreatment.” Simulated RMD-guided BL therapy would be active in 95% of cases with a mean DOOR-MAT score of 91.4 (95% CI, 91.2–91.7), and 7% of cases would be classified as overtreatment.ConclusionIn a large cohort of BSI patients where rates of BL resistance were low, we observed that empiric BL therapy, although highly effective, is of broader spectrum than necessary. RMD-guided therapy has the potential to reduce overtreatment without compromising effective therapy. DOOR-MAT provides a flexible framework for measuring appropriateness of therapy for BSI. Disclosures All authors: No reported disclosures.

1459. The Scope of Mycoplasma Pneumoniae Pneumonia Diagnosed by Multiplex Polymerase Chain Reaction Respiratory Viral Panel in Pediatric Patients in Hawaii

Background Mycoplasma pneumoniae pneumonia (MPP) is classically associated with an infection in older children with mild virulence in younger children. The multiplex polymerase chain reaction (PCR) respiratory viral panel (RVP) allows for diagnosis of multiple viruses and bacteria.MethodsA retrospective study was performed in patients 0–18 years old with positive MPP RVP from January 1, 2013 to June 30, 2017. Clinical cases of patients hospitalized with positive MPP testing by RVP PCR were reviewed for clinical, radiologic and laboratory data.ResultsA total of 3,621 RVPs were tested with 49 positive for MPP. In regard to age of patients, 27/49 (incidence 2.7%) positive for MPP were under 5 years old as compared with 22/49 (incidence 1%) between 5–18 years old. 75% of RVPs obtained were in patients under 5 years of age. Cough and fever were present for a mean of 8.3 and 7.6 days, respectively prior to RVP. Of the MPP positive patients, 21/49 patients (43%) were treated with scheduled although only 16 had a history of wheezing. Of the MPP positive patients, 38/48 patients had radiological findings of a pulmonary infiltrate (not perihilar) with 30/38 patients (79%) had bilateral infiltrates. Admission antimicrobial therapy was the following: 8 on no antibiotic, 21 on nonmacrolide, 11 macrolide and nonmacrolide, and 9 on macrolide therapy alone. Pediatric intensive care unit (PICU) admission occurred in 8 patients: 4 direct PICU admissions and 4 patients transferred from wards to PICU. All four PICU transfers had initially nonmacrolide therapy; 3 of 4 were under 5 years of age.ConclusionOver half of Pediatric MPP was diagnosed by rapid molecular diagnostics in patients under 5 years of age. Bilateral pulmonary infiltrates and new onset wheezing responsive to β agonists were commonly noted in patients who had MPP. A small subset of those younger patients required higher level of care after initial therapy with nonmacrolide therapy. While MPP has a lower incidence among younger children, the infection is not rare and can have a significant clinical impact. MPP should be considered in all patients, especially younger patients who are nonresponsive to treatment of community acquired pneumonia.Disclosures All authors: No reported disclosures.

2065. Whole Genome Sequencing for Antimicrobial Resistance Prediction in MRSA and VRE: A Real-world Application

BackgroundThe antimicrobial resistance (AMR) crisis represents a serious threat to public health and the healthcare economy. The impact of increasing AMR has resulted in concentrated efforts to increased rapid molecular diagnostics of AMRs. In combination with publicly available web-based AMR databases, whole-genome sequencing (WGS) offers the capacity for detection of antibiotic resistance genes with low turnaround times and is becoming increasingly affordable. Here we sought to examine concordance between WGS-based resistance prediction and phenotypic susceptibility testing results for prospectively collected VRE and MRSA clinical isolates using publicly-available tools.MethodsMRSA and VRE isolates were prospectively collected and underwent WGS at the University of Pittsburgh Medical Center (UPMC) between December 2016 and December 2017. Antibiotic-resistant gene content was assessed by uploading assembled contigs to ResFinder, NCBI betalactamase and CARD using a BLASTn.search. Routine susceptibility was performed by Microscan™. Concordance between genotypic and phenotypic as well as sensitivity, specificity, positive and negative predictive values methods were calculated for each antibiotic/organism combination, using the phenotypic results as the gold standard. In case of discordance between the methods, repeat susceptibility using disc diffusion results was performed and was then considered to be the gold standard method.ResultsPhenotypic susceptibility testing and WGS results were available for 109 and 105 unique MRSA and VRE isolates, respectively. Out of total of 1,058 isolate/antibiotic combinations overall concordance of WGS-web-based prediction with phenotypic susceptibility methods was 99.1% with a sensitivity, specificity, PPV, NPV of 98, 99.6, 99.5, and 98.3%, respectively. Specific concordance for MRSA isolates was 98.8%. with a sensitivity, specificity, PPV and NPV of 97.6, 99.8, 99.7, and 98.5% (Table 1), while concordance for VRE isolates was 99.3%, with a sensitivity, specificity, PPV and NPV of 98.6, 98.1, 99.1, and 97.2% (Table 2). ConclusionWGS is a reliable predicator of phenotypic resistance for both MRSA and VRE.Disclosures All authors: No reported disclosures.

Rapid Molecular Diagnostics to Inform Empiric Use of Ceftazidime/Avibactam and Ceftolozane/Tazobactam Against Pseudomonas aeruginosa: PRIMERS IV.

Overcoming β-lactam resistance in pathogens such as Pseudomonas aeruginosa is a major clinical challenge. Rapid molecular diagnostics (RMDs) have the potential to inform selection of empiric therapy in patients infected by P. aeruginosa. In this study, we used a heterogeneous collection of 197 P. aeruginosa that included multidrug-resistant isolates to determine whether 2 representative RMDs (Acuitas Resistome test and VERIGENE gram-negative blood culture test) could identify susceptibility to 2 newer β-lactam/β-lactamase inhibitor (BL-BLI) combinations, ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (TOL/TAZO). We found that the studied RMD platforms were able to correctly identify BL-BLI susceptibility (susceptibility sensitivity, 100%; 95% confidence interval [CI], 97%, 100%) for both BLs-BLIs. However, their ability to detect resistance to these BLs-BLIs was lower (resistance sensitivity, 66%; 95% CI, 52%, 78% for TOL/TAZO and 33%; 95% CI, 20%, 49% for CZA). The diagnostic platforms studied showed the most potential in scenarios where a resistance gene was detected or in scenarios where a resistance gene was not detected and the prevalence of resistance to TOL/TAZO or CZA is known to be low. Clinicians need to be mindful of the benefits and risks that result from empiric treatment decisions that are based on resistance gene detection in P. aeruginosa, acknowledging that such decisions are impacted by the prevalence of resistance, which varies temporally and geographically.

Nanoplasmonic optical antennas for life sciences and medicine

Surface plasmons — light-induced oscillations of electrons at the surface of nanoplasmonic metallic nanoparticles or nanostructures — can be used in a wide range of applications. Such nanoplasmonic optical antennas can be interfaced with biological systems to answer diverse questions in life sciences and to solve problems in translational medicine. In particular, nanoplasmonics provide insight and solutions for intracellular exploration, gene delivery and regulation, and rapid precision molecular diagnostics. In this Review, we examine the development of nanoplasmonic optical antennas for in vitro and in vivo applications. We evaluate the use of optical nanoplasmonic antennas for the optical detection of mRNA in living cells and for in vivo molecular imaging. We also discuss nanoplasmonic optical antennas for in vivo gene delivery and the optical control of gene circuits. Finally, we highlight the use of nanoplasmonic-based molecular diagnostic systems for ultrafast precision medicine. Nanoplasmonics have emerged as a promising technology for applications in life sciences and medicine. In this Review, we discuss the application of nanoplasmonic optical antennas for in vivo intracellular exploration, photonic gene delivery and regulation, and in vitro molecular diagnostics.

Invasive candidiasis.

Invasive candidiasis is an important health-care-associated fungal infection that can be caused by several Candida spp.; the most common species is Candida albicans, but the prevalence of these organisms varies considerably depending on geographical location. The spectrum of disease of invasive candidiasis ranges from minimally symptomatic candidaemia to fulminant sepsis with an associated mortality exceeding 70%. Candida spp. are common commensal organisms in the skin and gut microbiota, and disruptions in the cutaneous and gastrointestinal barriers (for example, owing to gastrointestinal perforation) promote invasive disease. A deeper understanding of specific Candida spp. virulence factors, host immune response and host susceptibility at the genetic level has led to key insights into the development of early intervention strategies and vaccine candidates. The early diagnosis of invasive candidiasis is challenging but key to the effective management, and the development of rapid molecular diagnostics could improve the ability to intervene rapidly and potentially reduce mortality. First-line drugs, including echinocandins and azoles, are effective, but the emergence of antifungal resistance, especially among Candida glabrata, is a matter of concern and underscores the need to administer antifungal medications in a judicious manner, avoiding overuse when possible. A newly described pathogen, Candida auris, is an emerging multidrug-resistant organism that poses a global threat.

Smartphone-Based Mobile Detection Platform for Molecular Diagnostics and Spatiotemporal Disease Mapping.

Rapid and quantitative molecular diagnostics in the field, at home, and at remote clinics is essential for evidence-based disease management, control, and prevention. Conventional molecular diagnostics requires extensive sample preparation, relatively sophisticated instruments, and trained personnel, restricting its use to centralized laboratories. To overcome these limitations, we designed a simple, inexpensive, hand-held, smartphone-based mobile detection platform, dubbed "smart-connected cup" (SCC), for rapid, connected, and quantitative molecular diagnostics. Our platform combines bioluminescent assay in real-time and loop-mediated isothermal amplification (BART-LAMP) technology with smartphone-based detection, eliminating the need for an excitation source and optical filters that are essential in fluorescent-based detection. The incubation heating for the isothermal amplification is provided, electricity-free, with an exothermic chemical reaction, and incubation temperature is regulated with a phase change material. A custom Android App was developed for bioluminescent signal monitoring and analysis, target quantification, data sharing, and spatiotemporal mapping of disease. SCC's utility is demonstrated by quantitative detection of Zika virus (ZIKV) in urine and saliva and HIV in blood within 45 min. We demonstrate SCC's connectivity for disease spatiotemporal mapping with a custom-designed website. Such a smart- and connected-diagnostic system does not require any lab facilities and is suitable for use at home, in the field, in the clinic, and particularly in resource-limited settings in the context of Internet of Medical Things (IoMT).

Isothermal Amplification of Long, Discrete DNA Fragments Facilitated by Single-Stranded Binding Protein

Isothermal amplification methods for detection of DNA and RNA targets have expanded significantly in recent years, promising a new wave of simple and rapid molecular diagnostics. Current isothermal methods result in the generation of short fragments (<150 base pairs) or highly branched long DNA products. Here we report the amplification of discrete target fragments of several kilobases at 37 °C from both double- and single-stranded circular template DNA using specific primer pairs. In contrast to existing methods, this amplification requires only the single-stranded DNA-binding protein gp32 from bacteriophage T4 and a strand-displacing DNA polymerase. In addition to the discrete amplicon products, this method also produces higher molecular weight products consisting of multiple repeated copies of the amplicon and template DNA. We demonstrate that two features of gp32 enable this amplification: a facilitation of primer strand invasion into double-stranded DNA, and a suppression of non-homologous primer annealing and nonspecific amplification. The ability presented here to produce long, discrete DNA products in an isothermal reaction extends the scope of isothermal amplification to enable more useful applications of these promising methods.

Molecular approaches for biosurveillance of the cucurbit downy mildew pathogen, Pseudoperonospora cubensis

Globalization has allowed for rapid movement of plant pathogens that threaten food security. Successful disease management largely depends on timely and accurate detection of plant pathogens causing epidemics. Thus, biosurveillance of epidemic plant pathogens such as Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew, is becoming a priority to prevent disease outbreaks and deploy successful control efforts. Next Generation Sequencing (NGS) facilitates rapid development of genomics resources needed to generate molecular diagnostics assays for P. cubensis. Having information regarding the presence or absence of the pathogen, amount of inoculum, crop risk, time to initiate fungicide applications, and effective fungicides to apply would significantly contribute to reducing losses to cucurbit downy mildew. In this article, we discuss approaches to identify unique loci for rapid molecular diagnostics using genomic data, to develop molecular diagnostic tools that discriminate economically important pathogen alleles (i.e. mating type and fungicide resistance), and how to use molecular diagnostics with current and future spore trap strategies for biosurveillance purposes of important downy mildew pathogens. The combined use of these technologies within the already existent disease management framework has the potential to improve disease control.

Same-day genomic and epigenomic diagnosis of brain tumors using real-time nanopore sequencing

Molecular classification of cancer has entered clinical routine to inform diagnosis, prognosis, and treatment decisions. At the same time, new tumor entities have been identified that cannot be defined histologically. For central nervous system tumors, the current World Health Organization classification explicitly demands molecular testing, e.g., for 1p/19q-codeletion or IDH mutations, to make an integrated histomolecular diagnosis. However, a plethora of sophisticated technologies is currently needed to assess different genomic and epigenomic alterations and turnaround times are in the range of weeks, which makes standardized and widespread implementation difficult and hinders timely decision making. Here, we explored the potential of a pocket-size nanopore sequencing device for multimodal and rapid molecular diagnostics of cancer. Low-pass whole genome sequencing was used to simultaneously generate copy number (CN) and methylation profiles from native tumor DNA in the same sequencing run. Single nucleotide variants in IDH1, IDH2, TP53, H3F3A, and the TERT promoter region were identified using deep amplicon sequencing. Nanopore sequencing yielded ~0.1X genome coverage within 6 h and resulting CN and epigenetic profiles correlated well with matched microarray data. Diagnostically relevant alterations, such as 1p/19q codeletion, and focal amplifications could be recapitulated. Using ad hoc random forests, we could perform supervised pan-cancer classification to distinguish gliomas, medulloblastomas, and brain metastases of different primary sites. Single nucleotide variants in IDH1, IDH2, and H3F3A were identified using deep amplicon sequencing within minutes of sequencing. Detection of TP53 and TERT promoter mutations shows that sequencing of entire genes and GC-rich regions is feasible. Nanopore sequencing allows same-day detection of structural variants, point mutations, and methylation profiling using a single device with negligible capital cost. It outperforms hybridization-based and current sequencing technologies with respect to time to diagnosis and required laboratory equipment and expertise, aiming to make precision medicine possible for every cancer patient, even in resource-restricted settings.

Electrochemical sensors for identifying pyocyanin production in clinical Pseudomonas aeruginosa isolates

In clinical practice, delays in obtaining culture results impact patient care and the ability to tailor antibiotic therapy. Despite the advancement of rapid molecular diagnostics, the use of plate cultures inoculated from swab samples continues to be the standard practice in clinical care. Because the inoculation culture process can take between 24 and 48h before a positive identification test can be run, there is an unmet need to develop rapid throughput methods for bacterial identification. Previous work has shown that pyocyanin can be used as a rapid, redox-active biomarker for identifying Pseudomonas aeruginosa in clinical infections. However, further validation is needed to confirm pyocyanin production occurs in all clinical strains of P. aeruginosa. Here, we validate this electrochemical detection strategy using clinical isolates obtained from patients with hospital-acquired infections or with cystic fibrosis. Square-wave voltammetric scans of 94 different clinical P. aeruginosa isolates were taken to measure the concentration of pyocyanin. The results showed that all isolates produced measureable concentrations of pyocyanin with production rates correlated with patient symptoms and comorbidity. Further bioinformatics analysis confirmed that 1649 genetically sequenced strains (99.9%) of P. aeruginosa possess the two genes (PhzM and PhzS) necessary to produce pyocyanin, supporting the specificity of this biomarker. Confirming the production of pyocyanin by all clinically-relevant strains of P. aeruginosa is a significant step towards validating this strategy for rapid, point-of-care diagnostics.

Correction for Evans et al., "Informing Antibiotic Treatment Decisions: Evaluating Rapid Molecular Diagnostics To Identify Susceptibility and Resistance to Carbapenems against Acinetobacter spp. in PRIMERS III".

Volume 55, no. 1, p. 134–144, 2017, [https://doi.org/10.1128/JCM.01524-16][1]. Table S1 in the supplemental material: Isolates PR-369 and PR-469 were mislabeled, and a column containing an alternate list of isolate names (ARLG IDs) was inadvertently omitted. Revised supplemental material is posted

Rapid Detection of Urinary Tract Infections via Bacterial Nuclease Activity

Rapid and accurate bacterial detection methods are needed for clinical diagnostic, water, and food testing applications. The wide diversity of bacterial nucleases provides a rich source of enzymes that could be exploited as signal amplifying biomarkers to enable rapid, selective detection of bacterial species. With the exception of the use of micrococcal nuclease activity to detect Staphylococcus aureus, rapid methods that detect bacterial pathogens via their nuclease activities have not beendeveloped. Here, we identify endonuclease I as a robust biomarker for E.coli and develop a rapid ultrasensitive assay that detects its activity. Comparison of nuclease activities of wild-type and nuclease-knockout E.coli clones revealed that endonuclease I is the predominant DNase in E.coli lysates. Endonuclease I is detectable by immunoblot and activity assays in uropathogenic E.coli strains. A rapid assay that detects endonuclease I activity in patient urine with an oligonucleotide probe exhibited substantially higher sensitivity for urinary tract infections than that reported for rapid urinalysis methods. The 3hr turnaround time is much shorter than that of culture-based methods, thereby providing a means for expedited administration of appropriate antimicrobial therapy. We suggest this approach could address various unmet needs for rapid detection of E.coli.

Sexually Transmitted Viral Infections

Sexually transmitted diseases (STD) of viral origin comprise the most prevalent STDs on earth. The impact of viral STDs is compounded because, while they are treatable, they are not curable. Symptoms of infection are dependent on the type of infecting virus and can range from immune deficiency to skin lesions, warts, and cancer. Because of advances in antiviral therapies and rapid molecular diagnostics, patients harbor long-term, yet manageable conditions with this group of STDs. <b>ABBREVIATIONS:</b>STD - Sexually Transmitted Disease, HIV - Human Immunodeficiency Virus, HSV - Herpes simplex virus, HPV - Human Papillomavirus, ssRNA - single stranded RNA, CDC - Center for Disease Control, NAT - Nucleic Acid Test, ART - Antiretroviral Therapy, NIH - National Institutes of Health, AIDS - Acquired Immunodeficiency Syndrome, CCR5 - C-C chemokine receptor, CSF - cerebral spinal fluid

Leading Antibacterial Laboratory Research by Integrating Conventional and Innovative Approaches: The Laboratory Center of the Antibacterial Resistance Leadership Group.

The Antibacterial Resistance Leadership Group (ARLG) Laboratory Center (LC) leads the evaluation, development, and implementation of laboratory-based research by providing scientific leadership and supporting standard/specialized laboratory services. The LC has developed a physical biorepository and a virtual biorepository. The physical biorepository contains bacterial isolates from ARLG-funded studies located in a centralized laboratory and they are available to ARLG investigators. The Web-based virtual biorepository strain catalogue includes well-characterized gram-positive and gram-negative bacterial strains published by ARLG investigators. The LC, in collaboration with the ARLG Leadership and Operations Center, developed procedures for review and approval of strain requests, guidance during the selection process, and for shipping strains from the distributing laboratories to the requesting investigators. ARLG strains and scientific and/or technical guidance have been provided to basic research laboratories and diagnostic companies for research and development, facilitating collaboration between diagnostic companies and the ARLG Master Protocol for Evaluating Multiple Infection Diagnostics (MASTERMIND) initiative for evaluation of multiple diagnostic devices from a single patient sampling event. In addition, the LC has completed several laboratory-based studies designed to help evaluate new rapid molecular diagnostics by developing, testing, and applying a MASTERMIND approach using purified bacterial strains. In collaboration with the ARLG's Statistical and Data Management Center (SDMC), the LC has developed novel analytical strategies that integrate microbiologic and genetic data for improved and accurate identification of antimicrobial resistance. These novel approaches will aid in the design of future ARLG studies and help correlate pathogenic markers with clinical outcomes. The LC's accomplishments are the result of a successful collaboration with the ARLG's Leadership and Operations Center, Diagnostics and Devices Committee, and SDMC. This interactive approach has been pivotal for the success of LC projects.