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Background: 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., Methods: 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., Results: 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%)., Conclusions: 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., Competing Interests: Potential conflicts of interest. P. J. S. reports grants and personal fees from Accelerate Diagnostics, OpGen Inc, bioMérieux Inc, BD Diagnostics, and Qiagen Sciences Inc; grants from Affinity Biosensors, Qiagen, Hardy Diagnostics, and T2 Diagnostics; personal fees from Roche Diagnostics, Shionogi, and GeneCapture; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from GenMark Dx, OpGen Inc, and BD Diagnostics; a leadership or fiduciary role on the Antibacterial Resistance Leadership Group Diagnostics Committee, CLSI AST Subcommittee, and CAP Microbiology Committee; stock options from GeneCapture, outside the submitted work. K. E. H. reports grants from BioFire Diagnostics and roles on the Infectious Diseases Society of America Board of Directors (CLIS Antifungal Susceptibility Committee), Clinical Infectious Diseases deputy editor, and JCM editor, outside the submitted work. R. P. reports grants from ContraFect, TenNor Therapeutics Limited, BioFire, and Adaptive Phage Therapeutics (paid to institution); is a consultant to Curetis, PathoQuest, Selux Diagnostics, 1928 Diagnostics, PhAST, Torus Biosystems, Day Zero Diagnostics, Mammoth Biosciences, HealthTrackRx, and Qvella (monies paid to Mayo Clinic; Mayo Clinic and R. P. have a relationship with Pathogenomix); has research supported by Adaptive Phage Therapeutics (Mayo Clinic has a royalty-bearing know-how agreement and equity in Adaptive Phage Therapeutics); is a consultant to Netflix, Abbott Laboratories, and CARB-X; has a 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; receives honoraria from the NBME, Up-to-Date, and the Infectious Diseases Board Review Course; reports travel reimbursement to author from ASM; and has a volunteer role on the Governance Committee of ASM. J. D. B. reports grants from Luminex Corporation, Abbott Molecular, Chromacode, and BioFire Diagnostics; is a consultant for Roche Diagnostics/GenMark, bioMérieux Inc, BioFire Diagnostics, and Qiagen; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from Thermo Fisher Scientific and Qiagen; payment for expert testimony from Lewis Rice; support for attending meetings and/or travel from Thermo Fisher Scientific; and a leadership or fiduciary role in other board, society, committee, or advocacy groups, paid or unpaid, with ASM and AMP. J. K. J. reports personal fees as speaker from BioFire Diagnostics. C. D. is a scientific advisor for GeneCapture and Quidel; is on the Shionogi speaker's bureau; has received travel funds from ASM; receives honoraria for editorial work performed for the Clinical Microbiology Newsletter; reports payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing, or educational events from ASM and NYASM; reports consulting fees from Quidel, Shionogi, Cepheid, and BD; and receipt of equipment, materials, drugs, medical writing, gifts, or other services from Accelerate. L. W. has received consulting fees from Roche Molecular Systems, Shionogi, and Talis Biomedical; research funding from Accelerate Diagnostics, bioMérieux Inc/BioFire Diagnostics, Hardy Diagnostics, and Roche Molecular Systems; and support for attending meetings and/or travel from the American Society for Microbiology (budget for speaking). G. Y. reports grants or contracts paid to institution from the Agency for Healthcare Research and Quality. All remaining authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The fusion (F) protein of respiratory syncytial virus (RSV) is the major target of immunoprophylactic monoclonal antibodies (mAbs) and vaccines. Recently reported mutations in F gene antigenic sites can vary among RSV types A and B. To further understand mutations in RSV F proteins, we performed subtyping and F gene sequencing on 400 RSV-positive respiratory samples collected at four pediatric hospitals within the United States from children under 2 years old between 2018 and 2020. RSV B was predominant in 2018-2019 and RSV A in 2019-2020 (55.5% and 85.5% respectively). Compared to the reference sequence, all RSV B samples had at least one antigenic polymorphism with the most changes at sites AM14/V (100%) and Ø (93.3%) followed by II (5.8%), IV (3.9%), and p27 (2.9%). The most frequent mutations among RSV B for AM14/V site were in L172Q (100%), S173L (100%), and K191R (95.2%) while for Ø site they were in I206M (93.3%) and Q209R (93.3%). Conversely, polymorphisms were observed in only 15.3% of RSV A samples overall, specifically at antigenic sites p27 (5.9%), IV (3.0%), II (2.5%), AM14/V (2.0%), I (2.0%), and Ø (0.5%). Among RSV A cases, T122A at p27 (n = 10) and S276N at II (n = 3) were the most common substitution sites. S276N at site II was found in both RSV types. Although polymorphisms in F proteins of RSV B were more common than those in RSV A samples, changes in both subtypes were observed in key F antigenic sites which could potentially impact the efficacy of mAb therapies and vaccines., (Copyright © 2022. Published by Elsevier B.V.)

SARS-CoV-2 was identified and diagnostic methods developed at an impressive speed due in great part to the wider use of molecular methods in 2019 compared with 2002 during the SARS pandemic. The development of rapid and novel molecular diagnostic assays, leveraging of the high adaptability of molecular tests, and the integration of SARS-CoV-2 genotyping into public health, clinical, and research laboratories have been some of the successes in SARS-CoV-2 molecular testing. The main challenges are related to regulatory hurdles, supply chain constraints, and laboratory preparation., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Urinary tract infections (UTIs) are among the most common bacterial infections in the United States and are a major driver of antibiotic use, both appropriate and inappropriate, across healthcare settings. Novel UTI diagnostics are a strategy that might enable better UTI treatment. Members of the Antibacterial Resistance Leadership Group Laboratory Center and the Infectious Diseases Society of America Diagnostics Committee convened to envision ideal future UTI diagnostics, with a view towards improving delivery of healthcare, patient outcomes and experiences, and antibiotic use, addressing which types of UTI diagnostics are needed and how companies might approach development of novel UTI diagnostics., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged into a world of maturing pathogen genomics, with more than 2 million genomes sequenced at the time of writing. The rise of more transmissible variants of concern that impact vaccine and therapeutic effectiveness has led to widespread interest in SARS-CoV-2 evolution. Clinicians are also eager to take advantage of the information provided by SARS-CoV-2 genotyping beyond surveillance purposes. Here, we review the potential role of SARS-CoV-2 genotyping in clinical care. The review covers clinical use cases for SARS-CoV-2 genotyping, methods of SARS-CoV-2 genotyping, assay validation and regulatory requirements, and clinical reporting for laboratories, as well as emerging issues in clinical SARS-CoV-2 sequencing. While clinical uses of SARS-CoV-2 genotyping are currently limited, rapid technological change along with a growing ability to interpret variants in real time foretells a growing role for SARS-CoV-2 genotyping in clinical care as continuing data emerge on vaccine and therapeutic efficacy.

Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae We collected 446 M. pneumoniae -positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required., (Copyright © 2020 Xiao et al.)

Background: In 2014, enterovirus D68 (EV-D68) was responsible for an outbreak of severe respiratory illness in children, with 1,153 EV-D68 cases reported across 49 states. Despite this, there is no commercial assay for its detection in routine clinical care. BioFire® Syndromic Trends (Trend) is an epidemiological network that collects, in near real-time, deidentified. BioFire test results worldwide, including data from the BioFire® Respiratory Panel (RP)., Objectives: Using the RP version 1.7 (which was not explicitly designed to differentiate EV-D68 from other picornaviruses), we formulate a model, Pathogen Extended Resolution (PER), to distinguish EV-D68 from other human rhinoviruses/enteroviruses (RV/EV) tested for in the panel. Using PER in conjunction with Trend, we survey for historical evidence of EVD68 positivity and demonstrate a method for prospective real-time outbreak monitoring within the network., Study Design: PER incorporates real-time polymerase chain reaction metrics from the RPRV/EV assays. Six institutions in the United States and Europe contributed to the model creation, providing data from 1,619 samples spanning two years, confirmed by EV-D68 gold-standard molecular methods. We estimate outbreak periods by applying PER to over 600,000 historical Trend RP tests since 2014. Additionally, we used PER as a prospective monitoring tool during the 2018 outbreak., Results: The final PER algorithm demonstrated an overall sensitivity and specificity of 87.1% and 86.1%, respectively, among the gold-standard dataset. During the 2018 outbreak monitoring period, PER alerted the research network of EV-D68 emergence in July. One of the first sites to experience a significant increase, Nationwide Children's Hospital, confirmed the outbreak and implemented EV-D68 testing at the institution in response. Applying PER to the historical Trend dataset to determine rates among RP tests, we find three potential outbreaks with predicted regional EV-D68 rates as high as 37% in 2014, 16% in 2016, and 29% in 2018., Conclusions: Using PER within the Trend network was shown to both accurately predict outbreaks of EV-D68 and to provide timely notifications of its circulation to participating clinical laboratories., (Published by Elsevier B.V.)

There are sparse data to indicate the extent that macrolide-resistant Mycoplasma pneumoniae (MRMp) occurs in the United States or its clinical significance. Between 2015 and 2018, hospitals in 8 states collected and stored respiratory specimens that tested positive for M. pneumoniae and sent them to the University of Alabama at Birmingham, where real-time PCR was performed for detection of 23S rRNA mutations known to confer macrolide resistance. MRMp was detected in 27 of 360 specimens (7.5%). MRMp prevalence was significantly higher in the South and East (18.3%) than in the West (2.1%). A2063G was the predominant 23S rRNA mutation detected. MICs for macrolide-susceptible M. pneumoniae (MSMp) were ≤0.008 μg/ml, whereas MICs for MRMp were 16 to 32 μg/ml. Patients with MRMp infection were more likely to have a history of immunodeficiency or malignancy. Otherwise, there were no other significant differences in the clinical features between patients infected with MRMp and those infected with MSMp, nor were there any differences in radiographic findings, hospitalization rates, viral coinfections, the mean duration of antimicrobial treatment, or clinical outcomes. There was no significant change in MRMp incidence over time or according to age, sex, race/ethnicity, or status as an inpatient or an outpatient. Patients with MRMp were more likely to have received a macrolide prior to presentation, and their treatment was more likely to have been changed to a fluoroquinolone after presentation. This is the first national surveillance program for M. pneumoniae in the United States. Additional surveillance is needed to assess the clinical significance of MRMp and to monitor changes in MRMp prevalence., (Copyright © 2019 American Society for Microbiology.)

Group A Streptococcus (GAS) is one of the leading causes of bacterial pharyngitis. Early GAS diagnosis is critical for appropriate antibiotic administration that reduces the risk of GAS sequelae and limits spread of the infection. The Aries Group A Strep (GAS) assay (Luminex, Austin, TX) is a fully automated PCR assay for direct detection of GAS in throat swab specimens in less than 2 h with minimum hands-on time. This multicenter prospective study evaluated the clinical performance of the Aries GAS assay compared to that of Streptococcus pyogenes culture. Subjects with symptoms consistent with pharyngitis were enrolled across four sites in the United States, and a throat swab in liquid Amies medium was obtained. Aries and reference testing was performed within 72 and 48 h after sample collection, respectively. Of 623 throat swab specimens from patients with pharyngitis (93.6% <18 years old, 54.3% female), the reference method yielded valid results for 618 specimens. Reference and Aries assay testing showed GAS-positive results for 160 (25.9%) and 166 (26.9%) specimens, respectively. Compared to the reference method, Aries assay sensitivity was 97.5% (95% confidence interval [CI], 93.7% to 99.0%), specificity was 97.8% (95% CI, 96.0 to 98.8%), positive predictive value was 94.0% (95% CI, 89.3% to 96.7%), and negative predictive value was 99.1% (95% CI, 97.7% to 99.7%). There were 10 false-positive and four false-negative detections with the Aries assay. Discrepant analysis with bidirectional sequencing yielded concordant results with the Aries assay for nine of 14 discordant samples. The Aries assay had high sensitivity and specificity for qualitative detection of group A Streptococcus from patients with pharyngitis., (Copyright © 2019 American Society for Microbiology.)

In pediatric practice it is common for infants under 2 months of age to undergo evaluation for sepsis when they are ill, often including lumbar puncture to assess for central nervous system (CNS) infection. The FilmArray Meningitis/Encephalitis (ME) panel is a newly approved test for rapid identification of CNS pathogens. Our objective was to study the epidemiology of CNS infection in young infants and the potential impact of rapid multiplex PCR on their care. A performance evaluation of the FilmArray ME panel was conducted from February 2014 to September 2014 at 11 sites. FilmArray ME panel results were compared to reference standards but not shared with providers. In our study, medical records for infants (aged 1 to 60 days) enrolled at three sites were reviewed for clinical, laboratory, and outcome data. A total of 145 infants were reviewed. The median age was 25 days. Most of the infants were hospitalized (134/145 [92%]) and received antibiotics (123/145 [85%]), and almost half (71/145 [49%]) received acyclovir. One infant had a bacterial pathogen, likely false positive, identified by the FilmArray ME panel. Thirty-six infants (25%) had a viral pathogen detected, including 21 enteroviruses. All infants with enteroviral meningitis detected by the FilmArray ME panel and conventional PCR were hospitalized, but 20% were discharged in less than 24 h when conventional PCR results became available. The FilmArray ME panel may play a role in the evaluation of young infants for CNS infection. Results may be used to guide management, possibly resulting in a decreased length of stay and less antimicrobial exposure for infants with low-risk viral infection detected., (Copyright © 2018 Blaschke et al.)

INTRODUCTIONSyndromic panels were first FDA cleared for detection of respiratory pathogens in 2008. Since then, other panels have been approved by the FDA, and most recently, the FilmArray meningitis/encephalitis panel (BioFire, Salt Lake City, UT) has become available. This assay detects 14 targets within 1 h and includes pathogens that typically cause different manifestations of infection, although they infect the same organ system. Several studies have reported both false-positive and false-negative results with this test, and all agree that the cost is significant. As with other panels, health care systems have adopted different strategies for offering this assay. Some have implemented strategies to limit the use of the test to certain patient populations, others have elected not to offer the test, and others have elected not to offer the test and instead request that providers order specific PCRs for the pathogens that best fit the patient's symptoms. In this Point-Counterpoint, Jennifer Dien Bard of the Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, and of the Keck School of Medicine at the University of Southern California explains why laboratories should offer these assays without restriction. Kevin Alby of the University of Pennsylvania explains the concerns about the use of these assays as first-line tests and why some limitations on their use might be appropriate., (Copyright © 2018 American Society for Microbiology.)

Due to the ongoing concern about the reliability of Staphylococcus breakpoints (interpretive criteria) for other β-lactam agents, the Clinical and Laboratory Standards Institute recently approved the elimination of all breakpoints for antistaphylococcal β-lactams except for penicillin, oxacillin or cefoxitin, and ceftaroline. Routine testing of penicillin and oxacillin or cefoxitin should be used to infer susceptibility for all β-lactams with approved clinical indications for staphylococcal infections. It is critical for laboratories to reject requests for susceptibility testing of other β-lactams against staphylococci and to indicate that susceptibility to these agents can be predicted from the penicillin and oxacillin or cefoxitin results. This article reviews β-lactam resistance mechanisms in staphylococci, current antimicrobial susceptibility testing and reporting recommendations for β-lactams and staphylococci, and microbiologic data and clinical data supporting the elimination of staphylococcal breakpoints for other β-lactam agents.