State of the Art: Acute Encephalitis.

Encephalitis is a devastating neurologic disease often complicated by prolonged neurologic deficits. Best practices for the management of adult patients include universal testing for a core group of etiologies, including herpes simplex virus (HSV)-1, varicella zoster virus (VZV), enteroviruses, West Nile virus, and anti-N-methyl-D-aspartate receptor (anti-NMDAR) antibody encephalitis. Empiric acyclovir therapy should be started at presentation and in selected cases continued until a second HSV-1 polymerase chain reaction test is negative. Acyclovir dose can be increased for VZV encephalitis. Supportive care is necessary for other viral etiologies. Patients in whom no cause for encephalitis is identified represent a particular challenge. Management includes repeat brain magnetic resonance imaging, imaging for occult malignancy, and empiric immunomodulatory treatment for autoimmune conditions. Next-generation sequencing (NGS) or brain biopsy should be considered. The rapid pace of discovery regarding autoimmune encephalitis and the development of advanced molecular tests such as NGS have improved diagnosis and outcomes. Research priorities include development of novel therapeutics.

Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Variants on Inpatient Clinical Outcome.

BACKGROUND: Prior observation has shown differences in COVID-19 hospitalization risk between SARS-CoV-2 variants, but limited information describes hospitalization outcomes. METHODS: Inpatients with COVID-19 at 5 hospitals in the eastern United States were included if they had hypoxia, tachypnea, tachycardia, or fever, and SARS-CoV-2 variant data, determined from whole-genome sequencing or local surveillance inference. Analyses were stratified by history of SARS-CoV-2 vaccination or infection. The average effect of SARS-CoV-2 variant on 28-day risk of severe disease, defined by advanced respiratory support needs, or death was evaluated using models weighted on propensity scores derived from baseline clinical features. RESULTS: Severe disease or death within 28 days occurred for 977 (29%) of 3369 unvaccinated patients and 269 (22%) of 1230 patients with history of vaccination or prior SARS-CoV-2 infection. Among unvaccinated patients, the relative risk of severe disease or death for Delta variant compared with ancestral lineages was 1.30 (95% confidence interval [CI]: 1.11-1.49). Compared with Delta, the risk for Omicron patients was .72 (95% CI: .59-.88) and compared with ancestral lineages was .94 (.78-1.1). Among Omicron and Delta infections, patients with history of vaccination or prior SARS-CoV-2 infection had half the risk of severe disease or death (adjusted hazard ratio: .40; 95% CI: .30-.54), but no significant outcome difference by variant. CONCLUSIONS: Although risk of severe disease or death for unvaccinated inpatients with Omicron was lower than with Delta, it was similar to ancestral lineages. Severe outcomes were less common in vaccinated inpatients, with no difference between Delta and Omicron infections.

Clinical Metagenomics for Infectious Diseases: Progress toward Operational Value.

The field of clinical metagenomics for infectious disease diagnostics has advanced to combining questions of technical methodologies with best-use practices due to lowering barriers of implementation. This commentary identifies challenges facing further development of the field and proposes methods for advancement by highlighting a recent prospective pilot study evaluating a targeted metagenomic approach for infectious endocarditis. This commentary introduces the concept of operational value as a method for standardizing results generated by differing clinical metagenomic approaches. Operational value includes assessments of result quality, utility, and cost through incorporating methodological aspects of metagenomics as applied to various infectious syndromes, patient populations, and specimen types. Focus is placed on standardizing outcome-based metrics using an operational value matrix. As ambitions of clinical metagenomics are increasingly realized, new models of study design and collaboration could promote progress toward routine use and positive benefits for patients with infectious diseases.

Infection With the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Delta Variant Is Associated With Higher Recovery of Infectious Virus Compared to the Alpha Variant in Both Unvaccinated and Vaccinated Individuals.

BACKGROUND: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC) B.1.617.2 (Delta) displaced B.1.1.7 (Alpha) and is associated with increases in coronavirus disease 2019 (COVID-19) cases, greater transmissibility, and higher viral RNA loads, but data are lacking regarding the infectious virus load and antiviral antibody levels in the nasal tract. METHODS: Whole genome sequencing, cycle threshold (Ct) values, infectious virus, anti-SARS-CoV-2 immunoglobulin G (IgG) levels, and clinical chart reviews were combined to characterize SARS-CoV-2 lineages circulating in the National Capital Region between January and September 2021 and differentiate infections in vaccinated and unvaccinated individuals by the Delta, Alpha, and B.1.2 (the predominant lineage prior to Alpha) variants. RESULTS: The Delta variant displaced the Alpha variant to constitute 99% of the circulating lineages in the National Capital Region by August 2021. In Delta infections, 28.5% were breakthrough cases in fully vaccinated individuals compared to 4% in the Alpha infected cohort. Breakthrough infections in both cohorts were associated with comorbidities, but only Delta infections were associated with a significant increase in the median days after vaccination. More than 74% of Delta samples had infectious virus compared to <30% from the Alpha cohort. The recovery of infectious virus with both variants was associated with low levels of local SARS-CoV-2 IgG. CONCLUSIONS: Infection with the Delta variant was associated with more frequent recovery of infectious virus in vaccinated and unvaccinated individuals compared to the Alpha variant but was not associated with an increase in disease severity in fully vaccinated individuals. Infectious virus was correlated with the presence of low amounts of antiviral IgG in the nasal specimens.

Genetic and Epidemiologic Analyses of an Outbreak of Pneumocystis jirovecii Pneumonia Among Kidney Transplant Recipients in the United States.

BACKGROUND: Pneumocystis jirovecii is an opportunistic fungus that causes Pneumocystis pneumonia (PCP) in immunocompromised hosts. Over an 11-month period, we observed a rise in cases of PCP among kidney-transplant recipients (KTR), prompting an outbreak investigation. METHODS: Clinical and epidemiologic data were collected for KTR diagnosed with PCP between July 2019 and May 2020. Pneumocystis strain typing was performed using restriction fragment length polymorphism analyses and multilocus sequence typing in combination with next-generation sequencing. A transmission map was drawn, and a case-control analysis was performed to determine risk factors associated with PCP. RESULTS: Nineteen cases of PCP in KTR were diagnosed at a median of 79 months post-transplantation; 8 received monthly belatacept infusions. Baseline characteristics were similar for KTR on belatacept versus other regimens; the number of clinic visits was numerically higher for the belatacept group during the study period (median 7.5 vs 3). Molecular typing of respiratory specimens from 9 patients revealed coinfection with up to 7 P. jirovecii strains per patient. A transmission map suggested multiple clusters of interhuman transmission. In a case-control univariate analysis, belatacept, lower absolute lymphocyte count, non-White race, and more transplant clinic visits were associated with an increased risk of PCP. In multivariate and prediction power estimate analyses, frequent clinic visits was the strongest risk factor for PCP. CONCLUSIONS: Increased clinic exposure appeared to facilitate multiple clusters of nosocomial PCP transmission among KTR. Belatacept was a risk factor for PCP, possibly by increasing clinic exposure through the need for frequent visits for monthly infusions.

A consensus conference to define the utility of advanced infectious disease diagnostics in solid organ transplant recipients.

The last decade has seen an explosion of advanced assays for the diagnosis of infectious diseases, yet evidence-based recommendations to inform their optimal use in the care of transplant recipients are lacking. A consensus conference sponsored by the American Society of Transplantation (AST) was convened on December 7, 2021, to define the utility of novel infectious disease diagnostics in organ transplant recipients. The conference represented a collaborative effort by experts in transplant infectious diseases, diagnostic stewardship, and clinical microbiology from centers across North America to evaluate current uses, unmet needs, and future directions for assays in 5 categories including (1) multiplex molecular assays, (2) rapid antimicrobial resistance detection methods, (3) pathogen-specific T-cell reactivity assays, (4) next-generation sequencing assays, and (5) mass spectrometry-based assays. Participants reviewed and appraised available literature, determined assay advantages and limitations, developed best practice guidance largely based on expert opinion for clinical use, and identified areas of future investigation in the setting of transplantation. In addition, attendees emphasized the need for well-designed studies to generate high-quality evidence needed to guide care, identified regulatory and financial barriers, and discussed the role of regulatory agencies in facilitating research and implementation of these assays. Findings and consensus statements are presented.

Evaluation of Metagenomic and Targeted Next-Generation Sequencing Workflows for Detection of Respiratory Pathogens from Bronchoalveolar Lavage Fluid Specimens.

Next-generation sequencing (NGS) workflows applied to bronchoalveolar lavage (BAL) fluid specimens could enhance the detection of respiratory pathogens, although optimal approaches are not defined. This study evaluated the performance of the Respiratory Pathogen ID/AMR (RPIP) kit (Illumina, Inc.) with automated Explify bioinformatic analysis (IDbyDNA, Inc.), a targeted NGS workflow enriching specific pathogen sequences and antimicrobial resistance (AMR) markers, and a complementary untargeted metagenomic workflow with in-house bioinformatic analysis. Compared to a composite clinical standard consisting of provider-ordered microbiology testing, chart review, and orthogonal testing, both workflows demonstrated similar performances. The overall agreement for the RPIP targeted workflow was 65.6% (95% confidence interval, 59.2 to 71.5%), with a positive percent agreement (PPA) of 45.9% (36.8 to 55.2%) and a negative percent agreement (NPA) of 85.7% (78.1 to 91.5%). The overall accuracy for the metagenomic workflow was 67.1% (60.9 to 72.9%), with a PPA of 56.6% (47.3 to 65.5%) and an NPA of 77.2% (68.9 to 84.1%). The approaches revealed pathogens undetected by provider-ordered testing (Ureaplasma parvum, Tropheryma whipplei, severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2], rhinovirus, and cytomegalovirus [CMV]), although not all pathogens detected by provider-ordered testing were identified by the NGS workflows. The RPIP targeted workflow required more time and reagents for library preparation but streamlined bioinformatic analysis, whereas the metagenomic assay was less demanding technically but required complex bioinformatic analysis. The results from both workflows were interpreted utilizing standardized criteria, which is necessary to avoid reporting nonpathogenic organisms. The RPIP targeted workflow identified AMR markers associated with phenotypic resistance in some bacteria but incorrectly identified blaOXA genes in Pseudomonas aeruginosa as being associated with carbapenem resistance. These workflows could serve as adjunctive testing with, but not as a replacement for, standard microbiology techniques.

Clinical implications of SARS-CoV-2 cycle threshold values in solid organ transplant recipients.

Detection of SARS-CoV-2 viral RNA by RT-PCR assays is the primary diagnostic test for COVID-19. Cycle threshold (CT ) values generated by some of these assays provide inversely proportional proxy measurements of viral load. The clinical implications of CT values are incompletely characterized, particularly in solid organ transplant (SOT) recipients. We conducted a retrospective chart review of 25 adult SOT recipients admitted to the Yale New Haven Health System between March 1 and May 15, 2020, analyzing 50 test results to investigate the clinical implications of SARS-CoV-2 CT values in this population. Initial CT values from upper respiratory tract samples were significantly higher in patients on tacrolimus, but were not associated with admission severity nor highest clinical acuity. Viral RNA was detected up to 38 days from symptom onset with a gradual increase in CT values over time. In five patients with serial testing, CT values <35.0 were detected >21 days after symptom onset in 4/5 and ≥27 days in 2/5, demonstrating prolonged RNA detection. These data describe SARS-CoV-2 viral dynamics in SOT patients and suggest that CT values may not be useful to predict COVID-19 severity in SOT patients. SARS-CoV-2 CT values may be more useful in informing infection prevention measures.

Diagnostic Stewardship for Next-Generation Sequencing Assays in Clinical Microbiology: An Appeal for Thoughtful Collaboration.

Next-generation sequencing (NGS)-based assays are primarily available from reference laboratories for diagnostic use. These tests can provide helpful diagnostic data but also can be overused by ordering providers not fully understanding their limitations. At present, there are few best practice guidelines for use. NGS-based assays can carry a high cost to institutions and individual patients, requiring thoughtful use through application of diagnostic stewardship principles. This article provides an overview of diagnostic stewardship approaches as applied to these assays, focusing on principles of collaboration, differential diagnosis formation, and seeking the best patient, syndrome, sample, timing, and test for improved patient care.

Re-emergence of Parechovirus: 2017-2022 National Trends of Detection in Cerebrospinal Fluid.

Increasing parechovirus (PeV) infections prompted a Centers for Disease Control and Prevention Health Advisory in July 2022. We retrospectively assessed national PeV trends in cerebrospinal fluid and observed unexpected viral dynamics from 2020 to 2022 with regional variance. These findings may be due to mitigation strategies aimed at severe acute respiratory syndrome coronavirus 2. PeV testing can benefit ill patients, particularly children.

False positive congenital cytomegalovirus saliva screening results with an FDA-approved assay at two institutions.

BACKGROUND: Congenital cytomegalovirus (CMV) infection is a significant cause of childhood hearing loss and developmental delay. Congenital CMV screening was implemented at two large hospital-affiliated laboratories using the FDA-approved Alethia CMV Assay Test System. In July 2022, an increase in suspected false-positive results was noted, leading to implementation of prospective quality management strategies. METHODS: The Alethia assay was performed per manufacturer-provided instructions on saliva swab specimens. After discovery of possible elevated false-positive rates, all positive results were confirmed by repeat Alethia testing on the same specimen, orthogonal polymerase chain reaction (PCR) on the same specimen, and/or clinical adjudication. Additionally, root cause analyses were conducted to pinpoint the source of false-positive results. RESULTS: At Cleveland Clinic (CCF), 696 saliva specimens were tested after initiation of the prospective quality management strategy, of which 36 (5.2%) were positive for CMV. Five of 36 (13.9%) were confirmed CMV positive by repeat Alethia testing and orthogonal PCR. Vanderbilt Medical Center (VUMC) tested 145 specimens, of which 11 (7.6%) were positive. Two of 11 (18.2%) confirmed as positive by orthogonal PCR or clinical adjudication. The remaining specimens (31 from CCF and 9 from VUMC) were negative for CMV by repeat Alethia and/or orthogonal PCR testing. DISCUSSION: These findings suggest a false positive rate of 4.5-6.2%, higher than the 0.2% reported for this assay in FDA claims. Laboratories using Alethia CMV may consider prospective quality management to evaluate all positive results. False-positive results can lead to unnecessary follow-up care and testing, and decreased confidence in laboratory testing.

Sequence Proven Reinfections with SARS-CoV-2 at a Large Academic Center.

Background: Increased reinfection rates with SARS-CoV-2 have recently been reported, with some locations basing reinfection on a second positive PCR test at least 90 days after initial infection. Methods: We identified cases where patients had two positive tests for SARS-CoV-2 and evaluated which of these had been sequenced as part of our surveillance efforts, and evaluated sequencing and clinical data. Results: 750 patients (920 samples) had a positive test at least 90 days after the initial test. The median time between tests was 377 days, and 724 (79%) of the post 90-day positives were collected after the emergence of the Omicron variant in November 2021. Sequencing was attempted on 231 samples and successful in 127. Successful sequencing spiked during the Omicron surge and showed higher median days from initial infection compared to failed sequences (median 398 days compared to 276 days, p<0.0005). A total of 122 (98%) patients showed evidence of reinfection, 45 of which had sequence proven reinfection and 77 had inferred reinfections (later sequence showed a clade that was not circulating when the patient was initially infected). Children accounted for only 4% of reinfections. 43 (96%) of 45 infections with sequence proven reinfection were caused by the Omicron variant, 41 (91%) were symptomatic, 32 (71%), were vaccinated prior to the second infection, and 6 (13%) were Immunosuppressed. Only 2 (4%) were hospitalized, and both had underlying conditions. Conclusion: Sequence proven reinfections increased with the Omicron variant but generally caused mild infections.