Correction: An amplification-free, 16S rRNA test for Neisseria gonorrhoeae in urine.

[This corrects the article DOI: 10.1039/D2SD00128D.].

An amplification-free, 16S rRNA test for Neisseria gonorrhoeae in urine.

An amplification-free, nanopore-based nucleic acid detection platform has been demonstrated for rapid, 16S rRNA sequence-specific detection of Neisseria gonorrhoeae at 10-100 CFU mL-1 in human urine against background bacterial flora at 1000 CFU mL-1. Gonorrhea is a very common notifiable communicable disease, antibiotic resistant strains have emerged, and the rate of reported gonococcal infections continues to increase. Since rapid clinical identification of bacterial pathogens in clinical samples is needed to guide proper antibiotic treatment and to control disease spread, it is important to engineer rapid, sensitive, selective, and inexpensive point-of-care (POC) diagnostic devices for pathogens such as N. gonorrhoeae. Our detector technology is based on straightforward conductometric detection of sustained blockage of a glass nanopore. Charge neutral, complementary peptide nucleic acid probes are conjugated to polystyrene beads to capture N. gonorrhoeae 16S rRNA selectively. In the presence of an electric field applied externally through a glass nanopore, the PNA-microbead conjugates that acquire substantial negative charge upon target hybridization are driven to the smaller diameter nanopore. At least partial blockage of the nanopore results in a sustained drop in ionic current that can be measured easily with simple electronics. The ability to detect N. gonorrhoeae over the range of 10 to 100 CFU mL-1 spiked in human urine was demonstrated successfully with estimated sensitivity and specificity of ∼98% and ∼100%, respectively. No false positives were observed for the control group of representative background flora (E. coli, K. pneumoniae, and E. faecalis) at 1000 CFU mL-1. Also, N. gonorrhoeae at 50 CFU mL-1 was successfully detected against 1000 CFU mL-1 of background flora in urine. These results suggest that this amplification-free technology may serve as the basis for rapid, inexpensive, low-power detection of pathogens in clinical samples at the POC.

Fungal Whole-Genome Sequencing for Species Identification: From Test Development to Clinical Utilization.

Using next-generation sequencing (NGS), we developed and validated a whole-genome sequencing (WGS)-based clinical test for fungal species identification on clinical isolates. The identification is mainly based on the fungal ribosomal internal transcribed spacer (ITS) region as the primary marker, and additional marker and genomic analysis applied for species within the Mucorales family (using the 28S rRNA gene) and Aspergillus genus (using the beta-tubulin gene and k-mer tree-based phylogenetic clustering). The validation study involving 74 unique fungal isolates (22 yeasts, 51 molds, and 1 mushroom-forming fungus) showed high accuracy, with 100% (74/74) concordance on the genus-level identifications and 89.2% (66/74) concordance on the species level. The 8 discrepant results were due to either the limitation of conventional morphology-based methodology or taxonomic changes. After one year of implementation in our clinical laboratory, this fungal NGS test was utilized in 29 cases; the majority of them were transplant and cancer patients. We demonstrated the utility of this test by detailing five case studies, in which accurate fungal species identification led to correct diagnosis, treatment adjustment or was ruled out for hospital acquired infection. This study provides a model for validation and implementation of WGS for fungal identification in a complex health system that serves a large immunocompromised patient population.

Ferrobotic swarms enable accessible and adaptable automated viral testing.

Expanding our global testing capacity is critical to preventing and containing pandemics1-9. Accordingly, accessible and adaptable automated platforms that in decentralized settings perform nucleic acid amplification tests resource-efficiently are required10-14. Pooled testing can be extremely efficient if the pooling strategy is based on local viral prevalence15-20; however, it requires automation, small sample volume handling and feedback not available in current bulky, capital-intensive liquid handling technologies21-29. Here we use a swarm of millimetre-sized magnets as mobile robotic agents ('ferrobots') for precise and robust handling of magnetized sample droplets and high-fidelity delivery of flexible workflows based on nucleic acid amplification tests to overcome these limitations. Within a palm-sized printed circuit board-based programmable platform, we demonstrated the myriad of laboratory-equivalent operations involved in pooled testing. These operations were guided by an introduced square matrix pooled testing algorithm to identify the samples from infected patients, while maximizing the testing efficiency. We applied this automated technology for the loop-mediated isothermal amplification and detection of the SARS-CoV-2 virus in clinical samples, in which the test results completely matched those obtained off-chip. This technology is easily manufacturable and distributable, and its adoption for viral testing could lead to a 10-300-fold reduction in reagent costs (depending on the viral prevalence) and three orders of magnitude reduction in instrumentation cost. Therefore, it is a promising solution to expand our testing capacity for pandemic preparedness and to reimagine the automated clinical laboratory of the future.

Genetic Predictive Factors for Nonsusceptible Phenotypes and Multidrug Resistance in Expanded-Spectrum Cephalosporin-Resistant Uropathogenic Escherichia coli from a Multicenter Cohort: Insights into the Phenotypic and Genetic Basis of Coresistance.

Antimicrobial resistance in urinary tract infections (UTIs) is a major public health concern. This study aims to characterize the phenotypic and genetic basis of multidrug resistance (MDR) among expanded-spectrum cephalosporin-resistant (ESCR) uropathogenic Escherichia coli (UPEC) causing UTIs in California patient populations. Between February and October 2019, 577 ESCR UPEC isolates were collected from patients at 6 clinical laboratory sites across California. Lineage and antibiotic resistance genes were determined by analysis of whole-genome sequence data. The lineages ST131, ST1193, ST648, and ST69 were predominant, representing 46%, 5.5%, 4.5%, and 4.5% of the collection, respectively. Overall, 527 (91%) isolates had an expanded-spectrum ß-lactamase (ESBL) phenotype, with blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaCTX-M-14 being the most prevalent ESBL genes. In the 50 non-ESBL phenotype isolates, 40 (62%) contained blaCMY-2, which was the predominant plasmid-mediated AmpC (pAmpC) gene. Narrow-spectrum ß-lactamases, blaTEM-1B and blaOXA-1, were also found in 44.9% and 32.1% of isolates, respectively. Among ESCR UPEC isolates, isolates with an ESBL phenotype had a 1.7-times-greater likelihood of being MDR than non-ESBL phenotype isolates (P < 0.001). The cooccurrence of blaCTX-M-15, blaOXA-1, and aac(6')-Ib-cr within ESCR UPEC isolates was strongly correlated. Cooccurrence of blaCTX-M-15, blaOXA-1, and aac(6')-Ib-cr was associated with an increased risk of nonsusceptibility to piperacillin-tazobactam, cefepime, fluoroquinolones, and amikacin as well as MDR. Multivariate regression revealed the presence of blaCTX-M-55, blaTEM-1B, and the ST131 genotype as predictors of MDR. IMPORTANCE The rising incidence of resistance to expanded-spectrum cephalosporins among Escherichia coli strains, the most common cause of UTIs, is threatening our ability to successfully empirically treat these infections. ESCR E. coli strains are often MDR; therefore, UTI caused by these organisms often leads to treatment failure, increased length of hospital stay, and severe complications (D. G. Mark, Y.-Y. Hung, Z. Salim, N. J. Tarlton, et al., Ann Emerg Med 78:357-369, 2021, https://doi.org/10.1016/j.annemergmed.2021.01.003). Here, we performed an in-depth analysis of genetic factors of ESCR E. coli associated with coresistance and MDR. Such knowledge is critical to advance UTI diagnosis, treatment, and antibiotic stewardship.

Clinical and epidemiological characteristics of SARS-CoV-2 Infection in Los Angeles County youth during the first year of the pandemic.

OBJECTIVES: The aim of this study was to characterize SARS-CoV-2 infection patterns in Los Angeles (LA) County youth followed at our institution during the first pandemic year. DESIGN: A prospective cohort of patients aged < 25 years who tested positive for SARS-CoV-2 using reverse-transcriptase polymerase chain reaction (RT-PCR) assays between March 13, 2020, and March 31, 2021, was evaluated at a large LA County health network. Demographics, age distribution, and disease severity were analyzed. RESULTS: There were 28,088 youth aged < 25 years tested for SARS-CoV-2 using RT-PCR, with 1849 positive results identified (7%). Among the positive results, 475 of 11,922 (4%) were identified at the pandemic onset (March-September 2020) (Cohort 1) and 1374 of 16,166 (9%) between October 2020 and March 2021 (Cohort 2), P < 0.001. When disease severity was compared across cohorts, Cohort 2 had a greater proportion of asymptomatic and mild/moderate disease categories than Cohort 1 (98% vs 80%, respectively); conversely, Cohort 1 had a near-10-fold higher proportion of severe disease than Cohort 2 (17% vs 1.8%). Cohort 2 comprised younger patients with a mean age of 13.7 years vs 17.3 years in Cohort 1. Older age was associated with a higher percentage of infection, with 63% of all confirmed cases found in participants aged 19 to 25 years in Cohort 1, compared with 38% of confirmed cases in Cohort 2. Age increase was also associated with greater disease severity by linear regression modeling (P< 0.001). CONCLUSION: Coronavirus disease 2019 (COVID-19) disease severity in youth decreased over time in LA County during the first pandemic year, likely a reflection of changing demographics, with younger children infected. A higher infection rate in youth did not lead to higher disease severity over time.

Genomic epidemiology of the Los Angeles COVID-19 outbreak and the early history of the B.1.43 strain in the USA.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused global disruption of human health and activity. Being able to trace the early outbreak of SARS-CoV-2 within a locality can inform public health measures and provide insights to contain or prevent viral transmission. Investigation of the transmission history requires efficient sequencing methods and analytic strategies, which can be generally useful in the study of viral outbreaks. METHODS: The County of Los Angeles (hereafter, LA County) sustained a large outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To learn about the transmission history, we carried out surveillance viral genome sequencing to determine 142 viral genomes from unique patients seeking care at the University of California, Los Angeles (UCLA) Health System. 86 of these genomes were from samples collected before April 19, 2020. RESULTS: We found that the early outbreak in LA County, as in other international air travel hubs, was seeded by multiple introductions of strains from Asia and Europe. We identified a USA-specific strain, B.1.43, which was found predominantly in California and Washington State. While samples from LA County carried the ancestral B.1.43 genome, viral genomes from neighboring counties in California and from counties in Washington State carried additional mutations, suggesting a potential origin of B.1.43 in Southern California. We quantified the transmission rate of SARS-CoV-2 over time, and found evidence that the public health measures put in place in LA County to control the virus were effective at preventing transmission, but might have been undermined by the many introductions of SARS-CoV-2 into the region. CONCLUSION: Our work demonstrates that genome sequencing can be a powerful tool for investigating outbreaks and informing the public health response. Our results reinforce the critical need for the USA to have coordinated inter-state responses to the pandemic.

Multicenter Clinical Evaluation of Vitek 2 Meropenem-Vaborbactam for Susceptibility Testing of Enterobacterales and Pseudomonas aeruginosa.

The carbapenem/beta-lactamase inhibitor meropenem-vaborbactam (MEV) used to treat complicated urinary tract infections and pyelonephritis in adults was approved in 2017 by the U.S. Food and Drug Administration (FDA). Here, we evaluated Vitek 2 MEV (bioMérieux, Durham, NC) compared to the reference broth microdilution (BMD) method. Of 449 Enterobacterales isolates analyzed per FDA/CLSI breakpoints, the overall performance was 98.2% essential agreement (EA), 98.7% category agreement (CA), and 0% very major errors (VME) or major errors (ME). For 438 FDA intended-for-use Enterobacterales isolates, performance was 98.2% EA, 98.6% CA, and 0% VME or ME. Evaluable EA was 81.0%, but with only 42 on-scale evaluable results. Individual species demonstrated EA and CA rates of ≥90% without any VME or ME. When evaluated using European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, overall Vitek 2 MEV performance for Enterobacterales and Pseudomonas aeruginosa demonstrated 97.3% EA, 99.2% CA, 2.3% VME, and 0.6% ME (after error resolution: 97.3% EA, 99.4% CA, 2.2% VME, and 0.4% ME) compared to the reference BMD method. Performance for P. aeruginosa included 92.2% EA, 97.4% CA, 0% VME, and 3.0% ME (after error resolution: 92.2% EA, 98.7% CA, 0% VME, and 1.5% ME). Performance for Enterobacterales included 98.2% EA, 99.6% CA, 3.0% VME, and 0.2% ME. Evaluable EA was 80.6% but was based on only 67 evaluable results. These findings support Vitek 2 MEV as an accurate automated system for MEV susceptibility testing of Enterobacterales and P. aeruginosa and could be an alternate solution to the manual-labor-intensive reference BMD method.

Case Report and Genomic Analysis of Cefiderocol-Resistant Escherichia coli Clinical Isolates.

OBJECTIVES: Cefiderocol is a novel siderophore cephalosporin with in vitro activity against multidrug-resistant (MDR), gram-negative bacteria and intrinsic structural stability to all classes of carbapenemases. We sought to identify gene variants that could affect the mechanism of action (MOA) of cefiderocol. METHODS: We report a case of bacteremia in a liver transplant candidate with a strain of carbapenem-resistant Escherichia coli that was found to be resistant to cefiderocol despite no prior treatment with this antimicrobial agent. Using whole-genome sequencing, we characterized the genomic content of this E coli isolate and assessed for genetic variants between related strains that were found to be cefiderocol susceptible. RESULTS: We identified several variants in genes with the potential to affect the mechanism of action of cefiderocol. CONCLUSIONS: The cefiderocol resistance in the E coli isolate identified in this study is likely due to mutations in the cirA gene, an iron transporter gene.

Retrospective Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in Symptomatic Patients Prior to Widespread Diagnostic Testing in Southern California.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused one of the worst pandemics in recent history. Few reports have revealed that SARS-CoV-2 was spreading in the United States as early as the end of January. In this study, we aimed to determine if SARS-CoV-2 had been circulating in the Los Angeles (LA) area at a time when access to diagnostic testing for coronavirus disease 2019 (COVID-19) was severely limited. METHODS: We used a pooling strategy to look for SARS-CoV-2 in remnant respiratory samples submitted for regular respiratory pathogen testing from symptomatic patients from November 2019 to early March 2020. We then performed sequencing on the positive samples. RESULTS: We detected SARS-CoV-2 in 7 specimens from 6 patients, dating back to mid-January. The earliest positive patient, with a sample collected on January 13, 2020 had no relevant travel history but did have a sibling with similar symptoms. Sequencing of these SARS-CoV-2 genomes revealed that the virus was introduced into the LA area from both domestic and international sources as early as January. CONCLUSIONS: We present strong evidence of community spread of SARS-CoV-2 in the LA area well before widespread diagnostic testing was being performed in early 2020. These genomic data demonstrate that SARS-CoV-2 was being introduced into Los Angeles County from both international and domestic sources in January 2020.

Predictors of SARS-CoV-2 Infection in Youth at a Large, Urban Healthcare Center in California, March-September 2020.

Objective: To understand which social, epidemiologic, and clinical risk factors are associated with SARS-CoV-2 infection in youth accessing care in a large, urban academic institution. Methods: We conducted a prospective cohort study with case-control analyses in youth who received testing for SARS-CoV-2 at our academic institution in Los Angeles during the first wave of the COVID-19 pandemic (March-September 2020). Results: A total of 27,976 SARS-CoV-2 assays among 11,922 youth aged 0-24 years were performed, including 475 youth with positive SARS-CoV-2 results. Positivity rate was higher among older, African American, and Hispanic/Latinx youth. Cases were more likely to be from non-English-speaking households and have safety-net insurance. Zip codes with higher proportion of Hispanic/Latinx and residents living under the poverty line were associated with increased SARS-CoV-2 cases. Youth were more likely to have positive results if tested for exposure (OR 21.5, 95% CI 14.6-32.1) or recent travel (OR 1.5, 95% CI 1.0-2.3). Students were less likely to have positive results than essential worker youth (OR 0.5, 95% CI 0.3-0.8). Patterns of symptom presentation varied significantly by age group; number of symptoms correlated significantly with age in SARS-CoV-2 cases (r = 0.030, p < 0.001). SARS-CoV-2 viral load did not vary by symptom severity, but asymptomatic youth had lower median viral load than those with symptoms (21.5 vs. 26.7, p = 0.009). Conclusions: Socioeconomic factors are important drivers of SARS-CoV-2 infection in youth. Presence of symptoms, exposure, and travel can be used to drive testing in older youth. Policies for school reopening and infection prevention should be tailored differently for elementary schools and universities.

Validation, Implementation, and Clinical Utility of Whole Genome Sequence-Based Bacterial Identification in the Clinical Microbiology Laboratory.

The application of next-generation sequencing extends from microbial identification to epidemiologic insight and antimicrobial resistance prediction. Despite this potential, the roadblock for clinical laboratories lies in implementation and validation of such complex technology and data analysis. Herein, a validation study used whole-genome sequencing (WGS) for pan-bacterial identification (ID) in a clinical laboratory, and assessed its clinical relevance. A diverse set of 125 bacterial isolates, including a subset without genus (25) and/or species (10) ID, were analyzed by de novo assembly and reference genome mapping. The 16S rRNA, rpoB, and groEL genes were used for ID. Using WGS, 100% (89 of 89) and 89% (79 of 89) of isolates were identified at the genus and species-levels, respectively. WGS also provided improved results for 71% (25/35) isolates originally reported with genus-only or descriptive IDs. Chart review identified cases in which improved genus and/or species-level ID by WGS may have had a positive impact on patient care. Reasons included the use of an ineffective antibiotic owing to unclear ID, use of antibiotics when not clinically indicated, and help with an outbreak investigation. The implementation of next-generation sequencing in a clinical microbiology setting is a challenging but necessary task. This study provides a model for the validation and implementation of bacterial ID by WGS in such a setting.

Massively scaled-up testing for SARS-CoV-2 RNA via next-generation sequencing of pooled and barcoded nasal and saliva samples.

Frequent and widespread testing of members of the population who are asymptomatic for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the mitigation of the transmission of the virus. Despite the recent increases in testing capacity, tests based on quantitative polymerase chain reaction (qPCR) assays cannot be easily deployed at the scale required for population-wide screening. Here, we show that next-generation sequencing of pooled samples tagged with sample-specific molecular barcodes enables the testing of thousands of nasal or saliva samples for SARS-CoV-2 RNA in a single run without the need for RNA extraction. The assay, which we named SwabSeq, incorporates a synthetic RNA standard that facilitates end-point quantification and the calling of true negatives, and that reduces the requirements for automation, purification and sample-to-sample normalization. We used SwabSeq to perform 80,000 tests, with an analytical sensitivity and specificity comparable to or better than traditional qPCR tests, in less than two months with turnaround times of less than 24 h. SwabSeq could be rapidly adapted for the detection of other pathogens.

Development and validation of a quantitative, non-invasive, highly sensitive and specific, electrochemical assay for anti-SARS-CoV-2 IgG antibodies in saliva.

Amperial™ is a novel assay platform that uses immobilized antigen in a conducting polymer gel followed by detection via electrochemical measurement of oxidation-reduction reaction between H2O2/Tetrametylbenzidine and peroxidase enzyme in a completed assay complex. A highly specific and sensitive assay was developed to quantify levels of IgG antibodies to SARS-CoV-2 in saliva. After establishing linearity and limit of detection we established a reference range of 5 standard deviations above the mean. There were no false positives in 667 consecutive saliva samples obtained prior to 2019. Saliva was obtained from 34 patients who had recovered from documented COVID-19 or had documented positive serologies. All of the patients with symptoms severe enough to seek medical attention had positive antibody tests and 88% overall had positive results. We obtained blinded paired saliva and plasma samples from 14 individuals. The plasma was analyzed using an EUA-FDA cleared ELISA kit and the saliva was analyzed by our Amperial™ assay. All 5 samples with negative plasma titers were negative in saliva testing. Eight of the 9 positive plasma samples were positive in saliva and 1 had borderline results. A CLIA validation was performed as a laboratory developed test in a high complexity laboratory. A quantitative non-invasive saliva based SARS-CoV-2 antibody test was developed and validated with sufficient specificity to be useful for population-based monitoring and monitoring of individuals following vaccination.

SARS-CoV-2 Infection Detection by PCR and Serologic Testing in Clinical Practice.

Patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be diagnosed by PCR during acute infection or later in their clinical course by detection of virus-specific antibodies. While in theory complementary, both PCR and serologic tests have practical shortcomings. A retrospective study was performed in order to further define these limitations in a clinical context and to determine how to best utilize these tests in a coherent fashion. A total of 3,075 patients underwent both PCR and serology tests at University of California, Los Angeles (UCLA), in the study period. Among these, 2,731 (89%) had no positive tests at all, 73 (2%) had a positive PCR test and only negative serology tests, 144 (5%) had a positive serology test and only negative PCR tests, and 127 (4%) had positive PCR and serology tests. Approximately half of the patients with discordant results (i.e., PCR positive and serology negative or vice versa) had mistimed tests in reference to the course of their disease. PCR-positive patients who were asymptomatic or pregnant were less likely to generate a detectable humoral immune response to SARS-CoV-2. On a quantitative level, the log number of days between symptom onset and PCR test was positively correlated with cycle threshold (CT) values. However, there was no apparent relationship between PCR CT and serologic (arbitrary units per milliliter) results.

Genomic Characterizations of Clade III Lineage of Candida auris, California, USA.

Candida auris is an emerging multidrug-resistant yeast. We describe an ongoing C. auris outbreak that began in October 2019 in Los Angeles, California, USA. We used genomic analysis to determine that isolates from 5 of 6 patients belonged to clade III; 4 isolates were closely related.

Swab-Seq: A high-throughput platform for massively scaled up SARS-CoV-2 testing.

The rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is due to the high rates of transmission by individuals who are asymptomatic at the time of transmission1,2. Frequent, widespread testing of the asymptomatic population for SARS-CoV-2 is essential to suppress viral transmission. Despite increases in testing capacity, multiple challenges remain in deploying traditional reverse transcription and quantitative PCR (RT-qPCR) tests at the scale required for population screening of asymptomatic individuals. We have developed SwabSeq, a high-throughput testing platform for SARS-CoV-2 that uses next-generation sequencing as a readout. SwabSeq employs sample-specific molecular barcodes to enable thousands of samples to be combined and simultaneously analyzed for the presence or absence of SARS-CoV-2 in a single run. Importantly, SwabSeq incorporates an in vitro RNA standard that mimics the viral amplicon, but can be distinguished by sequencing. This standard allows for end-point rather than quantitative PCR, improves quantitation, reduces requirements for automation and sample-to-sample normalization, enables purification-free detection, and gives better ability to call true negatives. After setting up SwabSeq in a high-complexity CLIA laboratory, we performed more than 80,000 tests for COVID-19 in less than two months, confirming in a real world setting that SwabSeq inexpensively delivers highly sensitive and specific results at scale, with a turn-around of less than 24 hours. Our clinical laboratory uses SwabSeq to test both nasal and saliva samples without RNA extraction, while maintaining analytical sensitivity comparable to or better than traditional RT-qPCR tests. Moving forward, SwabSeq can rapidly scale up testing to mitigate devastating spread of novel pathogens.

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

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: Rapid organism ID and phenotypic AST led to faster changes in antibiotic therapy for gram-negative BSIs. CLINICAL TRIALS REGISTRATION: NCT03218397.

Performance Characteristics of Severe Acute Respiratory Syndrome Coronavirus 2 RT-PCR Tests in a Single Health System: Analysis of >10,000 Results from Three Different Assays.

The current pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the approval of numerous molecular diagnostic assays with various performance and technical capacities. There are limited data comparing performance among assays. We conducted a retrospective analysis of >10,000 test results among three widely used RT-PCR assays for coronavirus disease 2019 (CDC, Simplexa Direct, and TaqPath) to assess performance characteristics. We also retested remnant weakly positive specimens to assess analytical sensitivity. All assays had strong linear correlation and little bias among CT values for PCR targets. In patients with first-test negative results (n = 811), most (795, 98.0%) remained negative for all subsequent testing. Retesting of weakly positive specimens (CT > 30) showed sensitivities as follows: TaqPath (97.8%), CDC (91%), Simplexa (75.3%). Our analysis showed no performance difference among PCR targets within the same assay, suggesting a single target is sufficient for SARS-CoV-2 detection. Lower respiratory tract specimens had a higher negative predictive value (100%) than upper respiratory tract specimens (98%), highlighting the utility of testing lower respiratory tract specimens when clinically indicated. Negative predictive value did not increase on further repeated testing, providing strong evidence for discouraging unnecessary repeated testing for SARS-CoV-2.

Carbapenem Resistant Aeromonas hydrophila Carrying blacphA7 Isolated From Two Solid Organ Transplant Patients.

Aeromonas hydrophila resides in a variety of aquatic environments. Infections with A. hydrophila mainly occur after contact with fresh or brackish water. Nosocomial infections with A. hydrophila can also occur. A. hydrophila infections can be difficult to treat due to both intrinsic and acquired antimicrobial resistance (AMR) mechanisms. In 2018-19, we isolated multi-drug resistant (MDR) A. hyrodphila from two solid organ transplant patients with intra-abdominal infections. We aimed to characterize their AMR mechanisms and to determine their genetic relatedness to aid epidemiological investigation. We performed whole genome sequencing (WGS) using Illumina MiSeq and Nanopore MinIon on 3 A. hydrophila isolates, with one isolate from Patient A (blood) and two isolates from Patient B (abdominal and T-tube fluid, isolated 2 weeks apart). Phenotypic assays included: Broth Microdilution (BMD), Modified Hodge Test (MHT), Modified Carbapenem Inactivation Method (mCIM), and EDTA Carbapenem Inactivation Method (eCIM). Data analyses were performed using CLCbio and Geneious. AMR genomic analysis revealed that all three isolates possess chromosomally encoded genes including blaOXA-12(oxacillinase), blacepS (AmpC), and blacphA7(metallo-beta-lactamase). All isolates tested strongly positive by MHT and mCIM, but only Patient B's second isolate (after 2 weeks of meropenem treatment) tested positive by eCIM. More intriguingly, Patient B's first isolate (before meropenem treatment) tested falsely susceptible to carbapenems by BMD, suggesting blacphA7 gene was not expressed constitutively. Phylogenetic analysis showed the two isolates from Patient B were highly similar with only 1 SNP difference. The isolate from Patient A only differed from Patient B's isolates by 35 and 36 SNPs, respectively, suggesting close genetic relatedness. Further epidemiological investigation is undergoing. We report the first cases of CphA-mediated carbapenem resistant A. hydrophila in the U.S. It is concerning that 1 out of 3 isolates tested falsely susceptible to carbapenems by BMD despite clear carbapenemase production shown by strongly positive MHT and mCIM. In both cases, meropenem was initially used to treat the patients. Clinicians and microbiologists in the US should be aware of the emerging MDR Aeromonas nosocomial infections and the potential false carbapenem susceptible results due to CphA-type carbapenemase, which may be induced during treatment.