Transmission, infectivity, and neutralization of a spike L452R SARS-CoV-2 variant.

We identified an emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant by viral whole-genome sequencing of 2,172 nasal/nasopharyngeal swab samples from 44 counties in California, a state in the western United States. Named B.1.427/B.1.429 to denote its two lineages, the variant emerged in May 2020 and increased from 0% to >50% of sequenced cases from September 2020 to January 2021, showing 18.6%-24% increased transmissibility relative to wild-type circulating strains. The variant carries three mutations in the spike protein, including an L452R substitution. We found 2-fold increased B.1.427/B.1.429 viral shedding in vivo and increased L452R pseudovirus infection of cell cultures and lung organoids, albeit decreased relative to pseudoviruses carrying the N501Y mutation common to variants B.1.1.7, B.1.351, and P.1. Antibody neutralization assays revealed 4.0- to 6.7-fold and 2.0-fold decreases in neutralizing titers from convalescent patients and vaccine recipients, respectively. The increased prevalence of a more transmissible variant in California exhibiting decreased antibody neutralization warrants further investigation.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Surveillance of Complicated Mpox Cases Unresponsive to Oral Tecovirimat in Los Angeles County, 2022.

The Los Angeles County Department of Public Health established a surveillance system to identify complicated (advanced human immunodeficiency virus [HIV] or hospitalized) mpox cases. From 1 August to 30 November 2022, we identified 1581 mpox cases, of which 134 (8.5%) were complicated. A subset of 8 cases did not recover after either initiating or completing a course of oral tecovirimat. All 8 patients were HIV positive and had advanced HIV (CD4 count <200 cells/µL). We identified 8 distinct mutations previously associated with tecovirimat resistance in specimens collected from 6 patients. Ongoing surveillance of viral evolution requires close coordination between health departments and frontline providers.

Investigation of a Suspect Severe Acute Respiratory Syndrome Coronavirus-2 and Influenza A Mixed Outbreak: Lessons Learned for Long-Term Care Facilities Nationwide.

A suspected outbreak of influenza A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at a long-term care facility in Los Angeles County was, months later, determined to not involve influenza. To prevent inadvertent transmission of infections, facilities should use highly specific influenza diagnostics and follow Centers for Disease Control and Prevention (CDC) guidelines that specifically address infection control challenges.

When Should Asymptomatic Persons Be Tested for COVID-19?

On 24 August 2020, the Centers for Disease Control and Prevention (CDC) updated its website to highlight that asymptomatic individuals, even those with exposure to a COVID-19-positive contact, do not necessarily need to be tested unless they have medical conditions associated with increased risk of severe illness from COVID-19. The CDC subsequently updated its guidance on 19 September 2020 to support testing of asymptomatic persons, including close contacts of persons with documented SARS-CoV-2 infection. In this editorial, the American Society for Microbiology Clinical and Public Health Microbiology Committee's Subcommittee on Laboratory Practices comments on testing of asymptomatic individuals relative to current medical knowledge of the virus and mitigation measures. Specific points are provided concerning such testing when undertaking contact tracing and routine surveillance. Limitations to consider when testing asymptomatic persons are covered, including the need to prioritize testing of contacts of positive COVID-19 cases. We urge the CDC to consult with primary stakeholders of COVID-19 testing when making such impactful changes in testing guidance.

Use of Molecular Epidemiology to Inform Response to a Hepatitis A Outbreak - Los Angeles County, California, October 2018-April 2019.

Los Angeles County comprises 4,058 square miles and is home to approximately 10 million residents (1), an estimated 59,000 (0.6%) of whom experience homelessness on a given night (2). In late 2018, Los Angeles County Department of Public Health (LAC DPH) was notified of a case of hepatitis A virus (HAV) infection in a person experiencing homelessness. LAC DPH conducted an investigation to determine the source of infection, identify additional cases, and identify contacts for postexposure prophylaxis (PEP). Over the next week, LAC DPH identified two additional hepatitis A cases in persons experiencing homelessness who knew one another socially and were known to congregate at a specific street intersection. To identify and respond rapidly to additional outbreak-associated cases, LAC DPH implemented enhanced surveillance procedures, including immediately obtaining specimens for molecular testing from all patients with suspected hepatitis A in the same geographic area. Enhanced surveillance identified four additional cases in persons linked to a senior living campus within two blocks of the intersection where the initial three patients reported congregating. These four cases were linked to the cluster in persons experiencing homelessness through HAV genotyping. Overall, DPH identified seven outbreak-associated hepatitis A cases during October 2018-January 2019. The DPH response to this community hepatitis A outbreak included conducting vaccination outreach to persons at risk, conducting environmental health outreach to restaurants in the outbreak area, and issuing health care provider alerts about the increased occurrence of hepatitis A. Implementation of near real-time molecular testing can improve hepatitis A outbreak responses by confirming HAV infections, linking additional cases to the outbreak, and informing the targeting of prevention efforts.

Fluoroquinolone Prophylaxis Selects for Meropenem-nonsusceptible Pseudomonas aeruginosa in Patients With Hematologic Malignancies and Hematopoietic Cell Transplant Recipients.

BACKGROUND: In Pseudomonas aeruginosa, fluoroquinolone exposure promotes resistance to carbapenems through upregulation of efflux pumps and transcriptional downregulation of the porin OprD. Evidence of this effect among hematologic malignancy (HM) patients or hematopoietic cell transplant (HCT) recipients receiving fluoroquinolone prophylaxis for neutropenia is lacking. METHODS: We retrospectively evaluated episodes of P. aeruginosa bloodstream infections in HM patients or HCT recipients over a 7-year period at our institution. We determined the association of fluoroquinolone prophylaxis at the time of infection with meropenem susceptibility of P. aeruginosa breakthrough isolates and risk factors for meropenem nonsusceptibility. Whole-genome sequencing (WGS) and phenotypic assessments of meropenem efflux pump activity were performed on select isolates to determine the mechanisms of meropenem resistance. RESULTS: We analyzed 55 episodes of P. aeruginosa bacteremia among 51 patients. Breakthrough bacteremia while on fluoroquinolone prophylaxis was associated with nonsusceptibility to meropenem, but not to antipseudomonal ß-lactams or aminoglycosides. The receipt of fluoroquinolone prophylaxis was independently predictive of bacteremia with a meropenem-nonsusceptible isolate. All meropenem-nonsusceptible isolates analyzed by WGS contained oprD inactivating mutations, and all meropenem-nonsusceptible isolates tested demonstrated reductions in the meropenem minimum inhibitory concentration in the presence of an efflux pump inhibitor. A phylogenetic analysis based on WGS revealed several clusters of closely related isolates from different patients. CONCLUSIONS: Fluoroquinolone prophylaxis in HM patients and HCT recipients is associated with breakthrough bacteremia with meropenem-nonsusceptible P. aeruginosa strains, likely due to both mutations increasing efflux pump activity and the epidemiology of P. aeruginosa bloodstream infections in our patient population.

Ceftazidime/avibactam resistance associated with L169P mutation in the omega loop of KPC-2.

OBJECTIVES: Ceftazidime/avibactam resistance due to mutation in the omega loop of KPC-2 has been documented in vitro and in vivo. This study evaluated the mechanism of ceftazidime/avibactam resistance in a KPC-2-expressing Klebsiella pneumoniae isolated from a patient following ceftazidime/avibactam combination therapy with gentamicin for the treatment of ventilator-associated pneumonia. METHODS: Ceftazidime/avibactam-susceptible and -resistant isolates of K. pneumoniae were evaluated by broth microdilution and WGS. The KPC-2 gene was cloned from the ceftazidime/avibactam-resistant isolate and evaluated for susceptibility to ceftazidime/avibactam, in an Escherichia coli background. RESULTS: A single L169P mutation was identified in the KPC-2 gene between the ceftazidime/avibactam-resistant and -susceptible isolates. The novel KPC-2 allele, designated KPC-35, was shown to confer reduced susceptibility to ceftazidime/avibactam and increased susceptibility to carbapenems, as compared with KPC-2. CONCLUSIONS: A novel L169P mutation was identified in KPC-2 and was shown through cloning experiments to confer reduced susceptibility to ceftazidime/avibactam.

The Frequency of Discordant Gyrase A Genotypes Among Cases of Multiple Neisseria gonorrhoeae Infections at Different Anatomic Sites.

Gyrase A genotyping reliably predicts Neisseria gonorrhoeae susceptibility to ciprofloxacin. It is unknown whether concurrent infections at different anatomic sites harbor different susceptibility profiles. We found a 3.2% frequency of discordant gyrase A genotypes among concurrent but anatomically separate N. gonorrhoeae infections diagnosed at 2 laboratories in Los Angeles.

Variability of Daptomycin MIC Values for Enterococcus faecium When Measured by Reference Broth Microdilution and Gradient Diffusion Tests.

Daptomycin has become a mainstay therapy for the treatment of serious vancomycin-resistant Enterococcus faecium infections. However, concern exists that current testing methods do not accurately predict the clinical success of daptomycin therapy. We evaluated a collection of 40 isolates of E. faecium across three centers by reference broth microdilution (BMD), and two gradient strips, to determine the precision of daptomycin MICs by these methods and the correlation of daptomycin MIC testing with mutations in the liaFSR system, one of the primary daptomycin resistance mechanisms among the enterococci. Daptomycin MICs spanned 3-log2 dilutions by BMD for 60.0% of isolates, 17.5% spanned 4 dilutions, 2.5% spanned 5 dilutions, and 20.0% spanned 6 or more dilutions. Fifteen isolates had MICs interpreted as susceptible by some tests and nonsusceptible by others. Neither BMD nor gradient diffusion tests could reliably differentiate isolates with or without mutations in liaFSR, resulting in a 59.8% very major error rate compared to determination of genotype by BMD, 63.5% by Etest, and 68.5% by MIC test strip. Imprecision in daptomycin MIC determination for E. faecium make establishment of a revised breakpoint challenging. Clinicians should be aware of this testing variability when making treatment decisions for patients with serious infections caused by this organism.

Selection of hyperproduction of AmpC and SME-1 in a carbapenem-resistant Serratia marcescens isolate during antibiotic therapy.

Objectives: Antibiotic selective pressure may result in changes to antimicrobial susceptibility throughout the course of infection, especially for organisms that harbour chromosomally encoded AmpC ß-lactamases, notably Enterobacter spp., in which hyperexpression of ampC may be induced following treatment with cephalosporins. In this study, we document a case of bacteraemia caused by a blaSME-1-harbouring Serratia marcescens that subsequently developed resistance to expanded-spectrum cephalosporins, piperacillin/tazobactam and fluoroquinolones, over the course of several months of treatment with piperacillin/tazobactam and ciprofloxacin. Methods: Susceptibility testing and WGS were performed on three S. marcescens isolates from the patient. ß-Lactamase activity in the presence or absence of induction by imipenem was measured by nitrocefin hydrolysis assays. Expression of ampC and blaSME-1 under the same conditions was determined by real-time PCR. Results: WGS demonstrated accumulation of missense and nonsense mutations in ampD associated with stable derepression of AmpC. Gene expression and ß-lactamase activity of both AmpC and SME-1 were inducible in the initial susceptible isolate, but were constitutively high in the resistant isolate, in which total ß-lactamase activity was increased by 128-fold. Conclusions: Although development of such in vitro resistance due to selective pressure imposed by antibiotics is reportedly low in S. marcescens, our findings highlight the need to evaluate isolates on a regular basis during long-term antibiotic therapy.

A Cost Analysis of Gyrase A Testing and Targeted Ciprofloxacin Therapy Versus Recommended 2-Drug Therapy for Neisseria gonorrhoeae Infection.

BACKGROUND: Novel approaches to combating drug-resistant Neisseria gonorrhoeae infections are urgently needed. Targeted therapy with ciprofloxacin has been made possible by a rapid assay for genotyping the gyrase A (gyrA) gene; a nonmutated gene reliably predicts susceptibility to ciprofloxacin. METHODS: We determined the costs of running the gyrA assay, 500 mg of ciprofloxacin, 250 mg of ceftriaxone injection, and 1000 mg of azithromycin. Cost estimates for gyrA testing included assay reagents and labor. Cost estimates for ceftriaxone included medication, injection, administration, supplies, and equipment. We measured the cost of using the gyrA assay and treatment based on genotype using previously collected data over a 13-month period between November 2015 and November 2016 for all N. gonorrhoeae cases diagnosed at UCLA. We subsequently developed 3 cost models, varying the frequency of testing and prevalence of N. gonorrhoeae infections with ciprofloxacin-resistant or genotype-indeterminate results. We compared those estimates with the cost of recommended 2-drug therapy (ceftriaxone and azithromycin). RESULTS: Based on a 65.3% prevalence of cases with ciprofloxacin-resistant or genotype indeterminate N. gonorrhoeae infections when running an average of 1.7 tests per day, the per-case cost of gyrA genotyping and targeted therapy was US $197.19. The per-case cost was US $155.16 assuming a 52.6% prevalence of ciprofloxacin-resistant or genotype-indeterminate infections when running an average of 17 tests per day. The per-case cost of 2-drug therapy was US $142.75. CONCLUSIONS: Direct costs of gyrA genotyping and targeted ciprofloxacin therapy depend on the prevalence of ciprofloxacin-resistant or genotype-indeterminate infections and testing frequency.

Implementation of a Rapid Genotypic Assay to Promote Targeted Ciprofloxacin Therapy of Neisseria gonorrhoeae in a Large Health System.

Multidrug-resistant Neisseria gonorrhoeae is a top threat to public health. In November 2015, UCLA Health introduced a rapid gyrase A (gyrA) genotypic assay for prediction of Neisseria gonorrhoeae susceptibility to ciprofloxacin. We found a significant reduction in ceftriaxone use with a concomitant increase in targeted therapy.

Evolution and Transmission of Carbapenem-Resistant Klebsiella pneumoniae Expressing the blaOXA-232 Gene During an Institutional Outbreak Associated With Endoscopic Retrograde Cholangiopancreatography.

BACKGROUND: Whole-genome sequencing (WGS) is an emerging and powerful technique by which to perform epidemiological studies in outbreak situations. METHODS: WGS was used to identify and evaluate an outbreak of OXA-232-expressing carbapenem-resistant Klebsiella pneumoniae (CRKP) transmitted to 16 patients over the course of 40 weeks via endoscopic retrograde cholangiopancreatography procedures at a single institution. WGS was performed on 32 OXA-232 CRKP isolates (1-7 per patient) and single-nucleotide variants (SNVs) were analyzed, with reference to the index patient's isolate. RESULTS: Interhost genetic diversity of isolates was between 0 and 15 SNVs during the outbreak; molecular clock calculations estimated 12.31 substitutions per genome per year (95% credibility interval, 7.81-17.05). Both intra- and interpatient diversification at the plasmid and transposon level was observed, significantly impacting the antibiogram of outbreak isolates. The majority of isolates evaluated (n = 27) harbored a blaCTX-M-15 gene, but some (n = 5) lacked the transposon carrying this gene, which resulted in susceptibility to aztreonam and third- and fourth-generation cephalosporins. Similarly, an isolate from a colonized patient lacked the transposon carrying rmtF and aac(6')lb genes, resulting in susceptibility to aminoglycosides. CONCLUSIONS: This study broadens the understanding of how bacteria diversify at the genomic level over the course of a defined outbreak and provides reference for future outbreak investigations.

Duodenoscope-Related Outbreak of a Carbapenem-Resistant Klebsiella pneumoniae Identified Using Advanced Molecular Diagnostics.

Background: Carbapenem-resistant Klebsiella pneumoniae infections are increasingly prevalent in North American hospitals. We describe an outbreak of carbapenem-resistant K. pneumoniae containing the blaOXA-232 gene transmitted by contaminated duodenoscopes during endoscopic retrograde cholangiopancreatography (ERCP) procedures. Methods: An outbreak investigation was performed when 9 patients with blaOXA-232 carbapenem-resistant K. pneumoniae infections were identified at a tertiary care hospital. The investigation included 2 case-control studies, review of duodenoscope reprocessing procedures, and culture of devices. Carbapenem-resistant Enterobacteriacieae (CRE) isolates were evaluated with polymerase chain reaction analysis for carbapenemase genes, and isolates with the blaOXA-232 gene were subjected to whole-genome sequencing and chromosome single-nucleotide polymorphism analysis. On recognition of ERCP as a key risk factor for infection, targeted patient notification and CRE screening cultures were performed. Results: Molecular testing ultimately identified 17 patients with blaOxa-232 carbapenem-resistant K. pneumoniae isolates, including 9 with infections, 7 asymptomatic carriers who had undergone ERCP, and 1 additional patient who had been hospitalized in India and was probably the initial carrier. Two case-control studies established a point-source outbreak associated with 2 specific duodenoscopes. A field investigation of the use, reprocessing, and storage of deuodenoscopes did not identify deviations from US Food and Drug Administration or manufacturer recommendations for reprocessing. Conclusions: This outbreak demonstrated the previously underappreciated potential for duodenoscopes to transmit disease, even after undergoing high-level disinfection according to manufacturers' guidelines.

Epidemiology of Neisseria gonorrhoeae Gyrase A Genotype, Los Angeles, California, USA.

We investigated the epidemiology of the mutant gyrase A gene, a reliable predictor of ciprofloxacin resistance, in Neisseria gonorrhoeae infections at UCLA Health in Los Angeles, California, USA, during November 1, 2015-August 31, 2016. Among 110 patients with N. gonorrhoeae infections, 48 (44%) had the mutant gyrase A gene.

Resistance to Ceftazidime-Avibactam Is Due to Transposition of KPC in a Porin-Deficient Strain of Klebsiella pneumoniae with Increased Efflux Activity.

Ceftazidime-avibactam is an antibiotic with activity against serine beta-lactamases, including Klebsiella pneumoniae carbapenemase (KPC). Recently, reports have emerged of KPC-producing isolates resistant to this antibiotic, including a report of a wild-type KPC-3 producing sequence type 258 Klebsiella pneumoniae that was resistant to ceftazidime-avibactam. We describe a detailed analysis of this isolate, in the context of two other closely related KPC-3 producing isolates, recovered from the same patient. Both isolates encoded a nonfunctional OmpK35, whereas we demonstrate that a novel T333N mutation in OmpK36, present in the ceftazidime-avibactam resistant isolate, reduced the activity of this porin and impacted ceftazidime-avibactam susceptibility. In addition, we demonstrate that the increased expression of blaKPC-3 and blaSHV-12 observed in the ceftazidime-avibactam-resistant isolate was due to transposition of the Tn4401 transposon harboring blaKPC-3 into a second plasmid, pIncX3, which also harbored blaSHV-12, ultimately resulting in a higher copy number of blaKPC-3 in the resistant isolate. pIncX3 plasmid from the ceftazidime-avibactam resistant isolate, conjugated into a OmpK35/36-deficient K. pneumoniae background that harbored a mutation to the ramR regulator of the acrAB efflux operon recreated the ceftazidime-avibactam-resistant MIC of 32 µg/ml, confirming that this constellation of mutations is responsible for the resistance phenotype.