Rapid Identification Of Organisms Research Articles

CRISPR-based diagnostics detects invasive insect pests.

Rapid identification of organisms is essential for many biological and medical disciplines, from understanding basic ecosystem processes, disease diagnosis, to the detection of invasive pests. CRISPR-based diagnostics offers a novel and rapid alternative to other identification methods and can revolutionize our ability to detect organisms with high accuracy. Here we describe a CRISPR-based diagnostic developed with the universal cytochrome-oxidase 1 gene (CO1). The CO1 gene is the most sequenced gene among Animalia, and therefore our approach can be adopted to detect nearly any animal. We tested the approach on three difficult-to-identify moth species (Keiferia lycopersicella, Phthorimaea absoluta and Scrobipalpa atriplicella) that are major invasive pests globally. We designed an assay that combines recombinase polymerase amplification (RPA) with CRISPR for signal generation. Our approach has a much higher sensitivity than real-time PCR assays and achieved 100% accuracy for identification of all three species, with a detection limit of up to 120 fM for P. absoluta and 400 fM for the other two species. Our approach does not require a sophisticated laboratory, reduces the risk of cross-contamination, and can be completed in less than 1 h. This work serves as a proof of concept that has the potential to revolutionize animal detection and monitoring.

Next generation marker-based vector concepts for rapid and unambiguous identification of single and double homozygous transgenic organisms.

For diploid model organisms, the actual transgenesis processes require subsequent periods of transgene management, which are challenging in emerging model organisms due to the lack of suitable methodology. We used the red flour beetle Tribolium castaneum, a stored-grain pest, to perform a comprehensive functional evaluation of our AClashOfStrings (ACOS) and the combined AGameOfClones/AClashOfStrings (AGOC/ACOS) vector concepts, which use four clearly distinguishable markers to provide full visual control over up to two independent transgenes. We achieved comprehensive statistical validation of our approach by systematically creating seventeen novel single and double homozygous sublines intended for fluorescence live imaging, including several sublines in which the microtubule cytoskeleton is labeled. During the mating procedures, we genotyped more than 20,000 individuals in less than 80 working hours, which corresponds to about 10 to 15 s per individual. We also confirm the functionality of our combined concept in two double transgene special cases, i.e. integration of both transgenes in close proximity on the same chromosome and integration of one transgene on the X allosome. Finally, we discuss our vector concepts regarding performance, genotyping accuracy, throughput, resource saving potential, fluorescent protein choice, modularity, adaptation to other diploid model organisms and expansion capability.

Application of MALDI-TOF Mass Spectrometry for the Assessment of Prevalence of Listeria monocytogenes in Raw Milk, Dairy Products and Freshwater Fishes

Background: Foodborne infections like Listeriosis cover several disorders and are a worldwide public health emergency. Listeria monocytogenes has been isolated from various foodstuffs, including milk and fishes. MALDI-TOF mass spectrometry technique is known for its rapid and accurate identification of bacterial organisms. Methods: In the current research, a total of 360 samples comprising raw milk (130), milk products (125) and freshwater fishes (105) were screened for the detection of pathogenic Listeria species by using the USDA method. The recovered Listeria isolates were characterized using conventional set of biochemical analysis along with sugar fermentation tests and further confirmed by MALDI-TOF MS. The virulent nature of pathogenic Listeria isolates was also assessed by in vitro tests like hemolysis on blood agar, CAMP and PI-PLC assay. Result: In current study, on screening 360 animal origin food samples, three isolates were recovered from raw milk samples and identified as Listeria monocytogenes indicating an overall prevalence of Listeria monocytogenes to the tune of 0.83%. Excellent correlation was observed with identification of Listeria species using conventional phenotypic tests and advanced molecular tool Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI-TOF MS) technique. The results depict dependability of advanced technique for rapid and reliable identification of Listeria species.

Diagnostic Accuracy of CHROMagar Orientation for Identification of Uropathogens

Objective: To determine the diagnostic accuracy of CHROMagar Orientation for its ability to support the growth and identification of uropathogens by keeping API 20E/20NE as a gold standard.
 Study Design: Cross-sectional study.
 Place and Duration of Study: Department of Microbiology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, from Jun to Nov 2019.
 Methodology: A total of 470 midstream specimen of urine from patients suspected of UTIs were analyzed. Urine specimens were inoculated by 1µL calibrated sterilized loop each on CLED Agar, Blood agar and CHROMagar Orientation media plates.Culture plates were incubated at 35.00±2.00ºC for 18 to 24 hours. As per standard protocols, isolates were identified by colony morphology, Gram stain, catalase, oxidase, DNAase, coagulase, and API 20E/20NE. In addition, the appearance of microorganism on CHROMagar Orientation media were identified according to the manufacturer’s instructions.
 Results: Out of 470 samples, 211 showed no growth, 90 were classified as mix growth (polymicrobial), and 169 samples were positive (unimicrobial) for the growth of uropathogens. All the samples positive for bacterial growth on conventional media were also positive on CHROMagar orientation and vice versa. The sensitivity and specificity of CHROMagar Orientation for differentiation of uropathogens were 99% and 100%, respectively.
 Conclusion: The overall findings of our study suggested that CHROMagar supports the growth of all uropathogens. It provided the rapid identification of organisms based on their colour morphology, even in a mixed bacterial culture, growth within 24 hours easily.

Integration of antimicrobial stewardship intervention with rapid organism identification improve outcomes in adult patients with bloodstream infections.

Integration of antimicrobial stewardship intervention (ASI) with rapid organism identification has the potential for early customization of antimicrobial therapy and improved clinical outcomes. We aimed to evaluate the impact of this combined approach on antimicrobial therapy-related outcomes in patients with bloodstream infections (BSIs). A pre-post quasi-experimental study was conducted to analyze the impact of ASI with organism identification via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) among patients with BSIs. Outcomes were compared to a historic pre-intervention group. The 30-day mortality was the primary endpoint. Secondary outcomes included time to first antibiotic modification, length of hospital stay. A total of 1004 adult patients with BSIs were included in the final analysis, 519 patients classified into the intervention group and 485 patients in the preintervention group. The patients in the intervention group were younger (66 vs. 70 years, P=0.02). The 30-day crude mortality (14.6% vs. 29.9%, P<0.001) was lower, the time to organism identification (72.25 vs. 83.6h, P<0.001) and length of hospital stay (12 days vs. 14 days, P<0.001) were shorter in the intervention group. Acceptance of an ASI was associated with a trend toward a reduced 30-day mortality on multivariable analysis (odds ratio 0.33; 95% CI: 0.24-0.47; P<0.001). The ASI combined with MALDI-TOF-MS approach decreased time to organism identification and time to appropriate antimicrobial therapy would achieve a better clinical outcome in the patients with BSIs.

Metagenomic Next-Generation Sequencing for Diagnosis of Pulmonary Infections

Despite pneumonia being a leading cause of morbidity and mortality worldwide, diagnostics remains a challenge, hindering rapid organism identification and subsequent effective treatments. Current microbiological methods include culture, serology, and limited molecular panels. While helpful, these methods are unable to address the full range of potential pathogens (e.g., fastidious or noncultivable organisms or uncommon organisms not included in current panels). Metagenomic next-generation sequencing (mNGS) is a molecular technique that analyzes and compares the nucleic acid content in a patient sample to a reference database of organisms that may include bacteria, viruses, fungi, and/or parasites, depending on the mNGS technology used. By bypassing the limitations of culture and targeted molecular assays, mNGS offers the potential to identify countless organisms directly from a patient specimen to aid in the diagnosis of an infectious process. Although promising, mNGS does have considerable limitations related to cost, interpretation, standardization, clinical relevance, turnaround time (TAT), and widespread availability. Thus, these factors should be considered prior to implementing mNGS for clinical use. Moreover, additional studies are required to fully understand the clinical and epidemiological impact of mNGS for the diagnosis of infectious diseases, including respiratory infections.

Reporting of Antimicrobial Resistance from Blood Cultures, an Antibacterial Resistance Leadership Group Survey Summary: Resistance Marker Reporting Practices from Positive Blood Cultures.

We assessed how laboratories use and handle reporting of results of rapid diagnostics performed on positive blood culture broths, with a focus on antimicrobial resistance (AMR) markers. A survey assembled by the Antibacterial Resistance Leadership Group Diagnostics Committee was circulated from December 2020 to May 2021. The survey was sent to local hospitals, shared on the ClinMicroNet and Division C listservs, and included in a College of American Pathologists proficiency testing survey. Ninety-six laboratories of various sizes across the United States (95%) and outside of the United States (5%) participated. Of the laboratories that had at least 1 rapid diagnostic in place (94%), significant heterogeneity in methods used and reporting practices was found across community (52%) and academic (40%) laboratories serving hospitals of various sizes. Respondents had implemented 1 to 6 different panels/platforms for a total of 31 permutations. Methods of reporting rapid organism identification and AMR results varied from listing all targets as "detected"/"not detected" (16-22%) without interpretive guidance, to interpreting results (23-42%), or providing therapeutic guidance comments to patient-facing healthcare teams (3-17%). Current approaches to reporting molecular AMR test results from positive blood culture vary significantly across clinical laboratories. Providing interpretative comments with therapeutic guidance alongside results reported may assist clinicians who are not well-versed in genetic mechanisms of AMR. However, this is currently not being done in all clinical laboratories. Standardized strategies for AMR gene result reporting are needed.

Recent Advances in Direct Blood Culture Phenotypic Antimicrobial Susceptibility Testing

Increasing rates of antibiotic resistance make selecting empiric therapy challenging for the treatment of sepsis and suspected bloodstream infections (BSIs). The time to initiation of effective therapy for BSIs is critical for positive patient outcomes. Recent advances in rapid diagnostics for the detection of BSIs directly from positive blood culture include rapid organism identification and rapid antimicrobial susceptibility testing (AST). Rapid AST methods include genotypic and phenotypic methods or a combination of both and provide important information to aid in the prompt initiation of effective therapy. Genotypic AST methods allow rapid direct detection of a resistance mechanism but may fail to accurately predict a full susceptibility profile, whereas phenotypic AST provides comprehensive results but is not rapid if conventional methods are used. Efforts to decrease the turnaround time of phenotypic AST are an important advancement for the treatment of BSIs. Here, we review currently available and in-development phenotypic methods for AST directly from positive blood culture and their potential benefits for antimicrobial stewardship and patient care.

Bacteremia in Pediatric Oncology Patients; A Single-Center Experience

Objective: To study organisms causing bacteremia and their outcome in cancer children with febrile neutropenia (FN) admitted at our centre.&#x0D; Study Design: Retrospective longitudinal study. &#x0D; Place and duration of study: Department of Pediatric Oncology, Shaukat Khanum Memorial Cancer Hospital, Lahore, from Feb to Dec 2017. &#x0D; Methodology: All pediatric oncology patients with febrile neutropenia admitted to the inpatient department were included. Data fields included age, diagnoses, demographics, organism types, time to positivity, multi-drug resistance, antibiotics, and outcome. &#x0D; Results: A total of 391 episodes of febrile neutropenia were documented among 86 patients. The mean age was 4.7±2.7 years. Twelve (14.0%) patients had intensive care admission, and 9(10.5%) of them died. Fifty-four (63.0%) children had primary diagnoses of haematological malignancies. Sixty-five percent had mucositis, central catheter, or both as risk factors. Thirty-nine isolates were cultured in 391 febrile-neutropenic episodes. Escherichia coli was the most frequently isolated organism in 16(41.0%) cultures, followed by Pseudomonas and Streptococcus pneumoniae in 4(10.3%) each. Poly-microbial isolates were seen in 6(15.4%) cultures. Multi-drug resistance was found in 12(30.8%) isolates. Thirty-four (87.0%) patients with positive cultures received appropriate antibiotics. Majority organisms were sensitive to Piperacillin/Tazobactam (14,35.9%) followed by Meropenem (10, 25.6%) and Colistin (6, 15.4%).&#x0D; Conclusions: Rapid identification of organisms from positive blood cultures combined with antimicrobial stewardship can have improved antibiotic treatment and outcomes.

P085 Prevalence and antifungal susceptibility of Candida spp. from the sputum sample of patients in a tertiary care hospital in Sikkim

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesThe role of Candida in sputum culture is unclear and is generally not treated when present in sputum samples. The objective of this study is to describe the clinical findings of patients with Candida spp. in sputum and their antifungal susceptibility pattern to know the local epidemiology of antifungal resistance.MethodsPatients with respiratory symptoms attending the tertiary care hospital during the study period of 6 months from June 2021 to December 2021. A total of 23 sputum samples were processed in the microbiology laboratory. Samples were processed following conventional mycological procedures including direct microscopy (visualization of budding yeast cell on grams stain), growth on sabouraud dextrose agar, Germ tube test, and CHROMagar. The isolates were identified by rapid identification (ID) of yeast and yeast-like organisms in the BD PhoenixTM Automated Microbiology System.Antifungal Susceptibility Testing (AFST) was carried out by disk diffusion susceptibility testing, Zone interpretation criteria as per M44/A2 protocol of CLSI were used. Antifungal used were amphotericin B (20 mcg), itraconazole (10 mcg), fluconazole (25 mcg), and voriconazole (1 mcg). Results of tests done on 23 isolates were collated and analyzed retrospectively.Clinical profile of the patients was taken retrospectively from record section and analyzed.ResultsOf the 23 patients, most common presentation was fever followed by cough and dyspnea. A total of 34% patients were receiving some form of steroid (injectable or inhalational). Only 2 patients were COVID positive by RT-PCR and 7(30%) patients had some radiological findings like consolidation, emphysematous changes, etc. Immunodeficiency condition was seen in 4 (17%) patients like tuberculosis and diabetes mellitus.Of the 23 samples, C. albicans showed prevalence of 91% as compared with C. tropicalis (5%) and C. glabrata (4.8%). AFST showed Candida spp. was found to be mostly sensitive to voriconazole and fluconazole. Resistance to amphotericin B was seen in most Candida spp. Itraconazole was not susceptible to even one isolate only 4 samples were intermediate (Fig. 1).ConclusionsInfections with Candida spp. are usually of low virulence and are associated with a few well-defined risk factors as immunocompromised state, malignancy, and steroid therapy. Understanding these risk factors, identifying the species with changing trends in antifungal resistance, instituting infection control practices to reduce morbidity and mortality in critical care areas can improve outcomes.Surveillance of the rates of Candida infection in critical areas, reporting of outbreaks and continuous monitoring of antifungal susceptibility patterns will help in choosing the best therapeutic management of complicated cases. Comparison of trends in infection rates amongst hospitals between various Indian cities and their resistance patterns can reveal vital information regarding the breakdown of infection control measures. Most Candida infections are of low virulence and only become significant in the vulnerable critical care areas. With the rise in prevalence of inherently azole-resistant species and rising use of echinocandins in ICUs, identifying risk factors and controlling the infection early can improve patient outcomes.

Improving outcomes and antibiotic stewardship (IOAS) for patients with Gram-positive bloodstream infections through use of rapid testing: a quasi-experimental multicentre study of the Accelerate PhenoTest™ BC Kit.

BackgroundData from the Improving Outcomes and Antibiotic Stewardship for Patients with Bloodstream Infections: Accelerate PhenoTest™ BC Kit (AXDX) Registry Study were analysed to determine the impact of rapid organism identification and antimicrobial susceptibility testing (AST) for Gram-positive bacteraemia.Patients and methodsThis multicentre, quasi-experimental study evaluated clinical and antimicrobial stewardship metrics following the implementation of AXDX. Data from hospitalized patients with bacteraemia were compared between groups, one that underwent testing on AXDX (post-AXDX) and one that underwent traditional identification and AST (pre-AXDX). An analysis of patients with Gram-positive bacteraemia was performed. The primary outcome was time to optimal therapy (TTOT). Secondary outcomes included time to first antibiotic modification (overall and Gram-positive), duration of unnecessary MRSA coverage, incidence of adverse events, length of stay and mortality.ResultsA total of 219 (109 pre-AXDX, 110 post-AXDX) patients with Gram-positive bacteraemia were included. Median TTOT was 36.3 h (IQR, 16.9–56.7) in the pre-AXDX group and 20.4 h (IQR, 7.5–36.7) in the post-AXDX group (P = 0.01). Compared with pre-AXDX, median time to first antibiotic modification (29.1 versus 15.9 h; P = 0.002), time to first Gram-positive antibiotic modification (33.2 versus 17.2 h; P = 0.003) and median duration of unnecessary MRSA coverage (58.4 versus 29.7 h; P = 0.04) were reduced post-AXDX. A trend towards decreased acute kidney injury (24% versus 13%; P = 0.06) was observed in the post-AXDX group. Groups did not differ in other secondary outcomes.ConclusionsImplementation of AXDX testing for patients with Gram-positive bacteraemia shortened the TTOT and reduced unnecessary antibiotic exposure due to faster antibiotic modifications.

Management of Pediatric Nonpathogenic Blood Cultures After Introduction of PCR Technology.

The rapid identification of organisms reported in positive blood cultures via polymerase chain reaction (PCR) can accurately identify a nonpathogenic bacterium and decrease time to definitive identification, as compared with traditional microbiologic methods. How this technology effects clinical and antimicrobial management in children with nonpathogenic bacteria identified in a blood culture without decision support has not been evaluated. A retrospective study of the management of children with positive blood culture results for nonpathogenic organisms before and after implementation of PCR technology. Each cohort's antibiotic management, frequency of repeat cultures, and return visits to an emergency department (ED) were compared. A total 136 patients during this time (49% [n = 67] pre-PCR and 51% [n = 69] post-PCR) had a blood culture positive for nonpathogenic bacterium. Admitted patients had a second specimen sent for testing on fewer occasions (P = .04); however, total antibiotic exposure did not differ significantly (P = .3) after introduction of PCR technology. There was no significant difference in length of stay postintervention (P = .12). Patients discharged directly from the ED had fewer return visits (P = .02) and received fewer repeat blood cultures (P = .04), and antibiotics were administered on fewer occasions after return (P = .04) postintroduction of PCR technology. With the addition of PCR technology, patients with blood cultures positive for nonpathogenic bacteria received less antibiotics, fewer repeat blood cultures, and fewer repeat ED evaluations.

Multiplexed Signal Ion Emission Reactive Release Amplification (SIERRA) Assay for the Culture-Free Detection of Gram-Negative and Gram-Positive Bacteria and Antimicrobial Resistance Genes.

The global prevalence of antibiotic-resistant bacteria has increased the risk of dangerous infections, requiring rapid diagnosis and treatment. The standard method for diagnosis of bacterial infections remains dependent on slow culture-based methods, carried out in central laboratories, not easily extensible to rapid identification of organisms, and thus not optimal for timely treatments at the point-of-care (POC). Here, we demonstrate rapid detection of bacteria by combining electrochemical immunoassays (EC-IA) for pathogen identification with confirmatory quantitative mass spectral immunoassays (MS-IA) based on signal ion emission reactive release amplification (SIERRA) nanoparticles with unique mass labels. This diagnostic method uses compatible reagents for all involved assays and standard fluidics for automatic sample preparation at POC. EC-IA, based on alkaline phosphatase-conjugated pathogen-specific antibodies, quantified down to 104 bacteria per sample when testing Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa lysates. EC-IA quantitation was also obtained for wound samples. The MS-IA using nanoparticles against S. aureus, E. coli, Klebsiella pneumoniae, and P. aeruginosa allowed selective quantitation of ∼105 bacteria per sample. This method preserves bacterial cells allowing extraction and amplification of 16S ribosomal RNA genes and antibiotic resistance genes, as was demonstrated through identification and quantitation of two strains of E. coli, resistant and nonresistant due to β-lactamase cefotaximase genes. Finally, the combined immunoassays were compared against culture using remnant deidentified patient urine samples. The sensitivities for these immunoassays were 83, 95, and 92% for the prediction of S. aureus, P. aeruginosa, and E. coli or K. pneumoniae positive culture, respectively, while specificities were 85, 92, and 97%. The diagnostic platform presented here with fluidics and combined immunoassays allows for pathogen isolation within 5 min and identification in as little as 15 min to 1 h, to help guide the decision for additional testing, optimally only on positive samples, such as multiplexed or resistance gene assays (6 h).

Impact of an Antimicrobial Stewardship Program-bundled initiative utilizing Accelerate Pheno\u2122 system in the management of patients with aerobic Gram-negative bacilli bacteremia

PurposeGram-negative bacteria (GNB) are a leading cause of bloodstream infections (BSI) and management is complicated by antibiotic resistance. The Accelerate Pheno™ system (ACC) can provide rapid organism identification and antimicrobial susceptibility testing (AST).MethodsA retrospective, pre-intervention/post-intervention study was conducted to compare management of non-critically ill patients with GNB BSI before and after implementation of a bundled initiative. This bundled initiative included dissemination of a clinical decision algorithm, ACC testing on all GNB isolated from blood cultures, real-time communication of results to the Antimicrobial Stewardship Program (ASP), and prospective audit with feedback by the ASP. The pre-intervention period was January 2018 through December 2018, and the post-intervention period was May 2019 through February 2020.ResultsSeventy-seven and 129 patients were included in the pre-intervention and post-intervention cohorts, respectively. When compared with the pre-intervention group, the time from Gram stain to AST decreased from 46.1 to 6.9 h (p < 0.001), and the time to definitive therapy (TTDT) improved from 32.6 to 10.5 h (p < 0.001). Implementation led to shorter median total duration of antibiotic therapy (14.2 vs 9.5 days; p < 0.001) and mean hospital length of stay (7.9 vs 5.3 days; p = 0.047) without an increase in 30-day readmissions (22.1% vs 14%; p = 0.13).ConclusionImplementation of an ASP-bundled approach incorporating the ACC aimed at optimizing antibiotic therapy in the management GNB BSI in non-critically ill patients led to reduced TTDT, shorter duration of antibiotic therapy, and shorter hospital length of stay without adversely affecting readmission rates.

Chip‐based duplex real‐time PCR for water quality monitoring concerning Legionella pneumophila and Legionella spp.

AbstractBased on biomolecular methods, rapid and selective identification of human pathogenic water organisms becomes an important issue. Legionella spp., are pathogenic water bacteria with worldwide significance. Prevalent detection methods for these microorganisms are time and/or cost intensive. We describe a detection setup and relating DNA assay. A miniaturized real‐time polymerase chain reaction (real‐time PCR) for direct on‐line discrimination of Legionella pneumophila and Legionella spp. was established and integrated into a real‐time PCR‐chip‐system. The PCR‐chip device combines a temperature controlling unit and a fluorescence intensity measurement. It was designed to achieve rapid amplification, using an approach of real‐time fluorescence read out with the intercalating dye EvaGreen® and melting curve analysis, without requiring multiple probes. The presented results exhibit reproducibility and good sensitivity, showing that the setup is suitable for robust, rapid and cost‐efficient detection and monitoring of a variety of Legionella spp.in urban water samples.

Randomized Trial Evaluating Clinical Impact of RAPid IDentification and Susceptibility Testing for Gram-negative Bacteremia: RAPIDS-GN.

Rapid blood culture diagnostics are of unclear benefit for patients with gram-negative bacilli (GNB) bloodstream infections (BSIs). We conducted a multicenter, randomized, controlled trial comparing outcomes of patients with GNB BSIs who had blood culture testing with standard-of-care (SOC) culture and antimicrobial susceptibility testing (AST) vs rapid organism identification (ID) and phenotypic AST using the Accelerate Pheno System (RAPID). Patients with positive blood cultures with Gram stains showing GNB were randomized to SOC testing with antimicrobial stewardship (AS) review or RAPID with AS. The primary outcome was time to first antibiotic modification within 72 hours of randomization. Of 500 randomized patients, 448 were included (226 SOC, 222 RAPID). Mean (standard deviation) time to results was faster for RAPID than SOC for organism ID (2.7 [1.2] vs 11.7 [10.5] hours; P < .001) and AST (13.5 [56] vs 44.9 [12.1] hours; P < .001). Median (interquartile range [IQR]) time to first antibiotic modification was faster in the RAPID arm vs the SOC arm for overall antibiotics (8.6 [2.6-27.6] vs 14.9 [3.3-41.1] hours; P = .02) and gram-negative antibiotics (17.3 [4.9-72] vs 42.1 [10.1-72] hours; P < .001). Median (IQR) time to antibiotic escalation was faster in the RAPID arm vs the SOC arm for antimicrobial-resistant BSIs (18.4 [5.8-72] vs 61.7 [30.4-72] hours; P = .01). There were no differences between the arms in patient outcomes. Rapid organism ID and phenotypic AST led to faster changes in antibiotic therapy for gram-negative BSIs. NCT03218397.

DNA Barcoding of Nematodes Using the MinION

Many nematode species are parasitic and threaten the health of plants and animals, including humans, on a global scale. Advances in DNA sequencing techniques have allowed for the rapid and accurate identification of many organisms including nematodes. However, the steps taken from sample collection in the field to molecular analysis and identification can take many days and depend on access to both immovable equipment and a specialized laboratory. Here, we present a protocol to genetically identify nematodes using 18S SSU rRNA sequencing using the MinION, a portable third generation sequencer, and proof that it is possible to perform all the molecular preparations on a fully portable molecular biology lab – the Bentolab. We show that both parasitic and free-living nematode species (Anisakis simplex, Panagrellus redivivus, Turbatrix aceti, and Caenorhabditis elegans) can be identified with a 96–100% accuracy compared to Sanger sequencing, requiring only 10–15 min of sequencing. This protocol is an essential first step toward genetically identifying nematodes in the field from complex natural environments (such as feces, soil, or marine sediments). This increased accessibility could in turn improve global information of nematode presence and distribution, aiding near-real-time global biomonitoring.

1062. Provider-Based Survey to Determine the Relative Importance of Clinical Factors Used to Make Empiric Antibiotic Decisions

BackgroundDespite advances in rapid organism identification, clinicians must make empiric antibiotic decisions prior to knowing the causative organism. Though many risk factors and patient characteristics have been studied and incorporated into predictive clinical decision support tools for empiric antibiotic prescription, little evidence exists on the relative importance of these factors in clinician judgment. The purpose of this study was to establish how providers evaluate patient and clinical risk factors when making empiric antimicrobial decisions.MethodsAn anonymous survey tool was distributed in February 2019 to medical providers who routinely prescribe antibiotics in 4 acute care hospitals throughout the University of Pennsylvania Health System. Providers were asked to rank the importance of 12 clinical factors using a 5-point scale from not important at all (1) to extremely important (5) in 3 unique clinical scenarios: uncomplicated cystitis, septic shock of unknown etiology, and uncomplicated pneumonia.ResultsResponse rate was 30% (217/732). The importance of each factor varied among different clinical scenarios except for prior antibiotic exposure, local resistance patterns, drug-drug interactions, and treatment guidelines. However, the absolute difference varied by the factor and clinical scenario. Presence of indwelling catheter(s) had the largest absolute variation (median difference of 2 between septic shock and pneumonia, P < 0.001), and it was one of the top 5 most important factors for septic shock, but the least important factor for pneumonia.ConclusionThe importance of clinicians place on clinical factors varies in different clinical scenarios. A better understanding of clinical decision-making in empiric antibiotic prescribing has the potential to guide stewardship efforts and clinical decision support. Disclosures All authors: No reported disclosures.

640. Randomized Clinical Trial Evaluating Clinical Impact of RAPid IDentification and Antimicrobial Susceptibility Testing for Gram-Negative Bacteremia (RAPIDS-GN)

BackgroundRapid blood culture diagnostics increase cost and have unclear benefit for patients with Gram-negative bacilli (GNB) bloodstream infections (BSIs). We conducted a multicenter, prospective randomized controlled trial (RAPIDS-GN), comparing outcomes of patients with GNB BSI who had blood culture testing with standard of care (SOC) culture and antibiotic susceptibility testing (AST) vs. rapid organism identification (ID) and phenotypic AST using the Accelerate Pheno System (AXDX).MethodsSubjects with blood culture Gram stain showing GNB were randomized to receive SOC testing with antimicrobial stewardship review (AS) or AXDX plus SOC testing with AS, at two academic medical centers between October 2017 and October 2018. SOC testing included rapid MALDI-TOF mass spectrometry ID and agar dilution or broth microdilution AST. In a modified intention to treat analysis, subjects were excluded if: Gram stain was erroneous, culture was positive during off-hours, blood culture in the prior week had GNB, they were deceased/on comfort care, or admitted to a nonparticipating hospital. The primary outcome was time to first antibiotic modification within 72 hours after randomization. Subjects without antibiotic modifications were assigned a time of 72 hours. No censoring was observed. T-tests and Wilcoxon rank-sum tests were used for statistical analyses.ResultsOf 500 randomized subjects, 448 were included (226 SOC, 222 AXDX). Groups did not differ in baseline characteristics (Table 1). Median (IQR) hours to first antibiotic modification was faster in the AXDX vs. SOC group [8.6 (2.6, 27.6) vs. 14.9 (3.3, 41.1)], P = 0.02 (Figure 1). Median (IQR) hours to first Gram-negative antibiotic modification (including escalation and de-escalation) was faster in the AXDX than SOC group [17.4 (4.9, 72) vs. 42.1 (10.1, 72)], P < 0.001 (Figure 2). Groups did not differ in clinical outcomes (Table 2). Mean (S.D.) time to results was faster for AXDX than SOC for organism ID [2.7 (1.2) h vs. 15.6 (20.3) h, P < 0.001] and AST [13 (55.7) h vs. 54.6 (45.5) h, P < 0.001].ConclusionIn the largest trial to evaluate the clinical impact of a blood culture diagnostic for GNB BSI, we found that rapid organism ID and phenotypic AST led to faster changes in antibiotic therapy for Gram-negative bacteremia. DisclosuresRitu Banerjee, MD, PhD, Accelerate Diagnostics: Grant/Research Support; BioFire: Research Grant; Biomerieux: Research Grant; Roche: Research Grant Robin Patel, MD, ASM and IDSA: Other Financial or Material Support, Travel reimbursement, editor’s stipends; CD Diagnostics, Merck, Hutchison Biofilm Medical Solutions, Accelerate Diagnostics, ContraFect, TenNor Therapeutics Limited, Shionogi: Grant/Research Support; Curetis, Specific Technologies, NextGen Diagnostics, PathoQuest, Qvella: Consultant; NBME, Up-to-Date, the Infectious Diseases Board Review Course: Honorarium recipient, Other Financial or Material Support; Patent on Bordetella pertussis/parapertussis PCR issued, a patent on a device/method for sonication with royalties paid by Samsung to Mayo Clinic, and a patent on an anti-biofilm substance issued: Other Financial or Material Support, Patents.

160. Could Reducing Time to Bacterial Identification From Positive Blood Cultures Improve Outcomes in Bacteremic Patients?

BackgroundSurvival of patients with septic shock is dependent on the timing of effective antibiotic administration. The initial notification by the microbiology lab of a positive blood culture is a key factor in improving patient outcomes. It can take >24 hours to definitively identify bacteria from positive blood cultures. Accordingly, we employed rapid organism identification and studied the impact of this on patient management from a quality improvement perspective.MethodsRapid organism identification was performed for bacteremic patients admitted to an ICU at St. Michael’s Hospital in Toronto, ON, by creating a pellet from positive blood culture bottles using a lysis centrifugation technique. MALDI-TOF was then used to obtain an organism identification. The microbiology lab verbally notified the ward clerk of the identification and surveys were conducted with treating physicians within 24–48 hours to evaluate the downstream impact of the rapid identification including changes to antibiotics, diagnostic testing, central line management and requests for specialty consultations.ResultsBetween January 28 and April 28, 2019, 17 rapid blood culture results were included for study. When asked how physicians received the result, in 7 cases the physician did not remember; other responses included microbiology report (2), nurse (2), pharmacist (1), antimicrobial stewardship or lab (1), on-call team (1) and residents (1). Antibiotics were adjusted in 13 patients; 3 of which may have changed antibiotics for reasons other than the organism identification. Reasons for not changing therapy include: appropriate empiric treatment, likely contaminants, or physician not being notified of the result. In 5 cases, all antibiotics were discontinued, in another 2 cases the antibiotics were broadened and a further 5 narrowed to cover the organism; the remaining 5 continued the same empiric therapy. Repeat blood cultures were obtained for 5 cases, follow-up imaging in 5 cases and lines were changed/removed in 5 cases. Consultation was requested for 7 cases.ConclusionBased on preliminary data, rapid organism identification shows promise of improved patient management with line removal and antibiotics adjustments occurring 1 day sooner with rapid results.Disclosures All authors: No reported disclosures.