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.

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.

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.

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.

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.

SARS-CoV-2 reinfections during the Delta and Omicron waves.

BACKGROUNDIncreased SARS-CoV-2 reinfection rates have been reported recently, with some locations basing reinfection on a second positive PCR test at least 90 days after initial infection. In this study, we used Johns Hopkins SARS-CoV-2 genomic surveillance data to evaluate the frequency of sequencing-validated, confirmed, and inferred reinfections between March 2020 and July 2022.METHODSPatients who had 2 or more positive SARS-CoV-2 tests in our system, with samples sequenced as a part of our surveillance efforts, were identified as the cohort for our study. SARS-CoV-2 genomes of patients' initial and later samples were compared.RESULTSA total of 755 patients (920 samples) had a positive test at least 90 days after the initial test, with a median time between tests of 377 days. Sequencing was attempted on 231 samples and was successful in 127. Rates of successful sequencing spiked during the Omicron surge; there was a higher median number of days from initial infection in these cases compared with those with failed sequences. A total of 122 (98%) patients showed evidence of reinfection; 45 of these patients had sequence-validated reinfection and 77 had inferred reinfections (later sequencing showed a clade that was not circulating when the patient was initially infected). Of the 45 patients with sequence-validated reinfections, 43 (96%) had reinfections that were caused by the Omicron variant, 41 (91%) were symptomatic, 32 (71%) were vaccinated prior to the second infection, 6 (13%) were immunosuppressed, and only 2 (4%) were hospitalized.CONCLUSIONSequence-validated reinfections increased with the Omicron surge but were generally associated with mild infections.FUNDINGFunding was provided by the Johns Hopkins Center of Excellence in Influenza Research and Surveillance (HHSN272201400007C), CDC (75D30121C11061), Johns Hopkins University President's Fund Research Response, Johns Hopkins Department of Pathology, and the Maryland Department of Health.

Combination Therapy With Casirivimab/Imdevimab and Remdesivir for Protracted SARS-CoV-2 Infection in B-cell-Depleted Patients.

Profoundly B-cell-depleted patients can have prolonged severe acute respiratory syndrome coronavirus 2 infections with evidence of active viral replication, due to inability to mount an adequate humoral response to clear the virus. We present 3 B-cell-depleted patients with prolonged coronavirus disease 2019 infection who were successfully treated with a combination of casirivimab/imdevimab and remdesivir.

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.

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.

The displacement of the SARS-CoV-2 variant Delta with Omicron: An investigation of hospital admissions and upper respiratory viral loads.

BACKGROUND: The increase in SARS-CoV-2 infections in December 2021 was driven primarily by the Omicron variant, which largely displaced the Delta over a three-week span. Outcomes from infection with Omicron remain uncertain. We evaluated whether clinical outcomes and viral loads differed between Delta and Omicron infections during the period when both variants were co-circulating. METHODS: In this retrospective observational cohort study, remnant clinical specimens, positive for SARS-CoV-2 after standard of care testing at the Johns Hopkins Microbiology Laboratory, between the last week of November and the end of December 2021, were used for whole viral genome sequencing. Cycle threshold values (Ct) for viral RNA, the presence of infectious virus, and levels of respiratory IgG were measured, and clinical outcomes were obtained. Differences in each measure were compared between variants stratified by vaccination status. FINDINGS: The Omicron variant displaced Delta during the study period and constituted 95% of the circulating lineages by the end of December 2021. Patients with Omicron infections (N = 1,119) were more likely to be vaccinated compared to patients with Delta (N = 908), but were less likely to be admitted (0.33 CI 0.21-0.52), require ICU level care (0.38 CI 0.17-0.87), or succumb to infection (0.26 CI 0.06-1.02) regardless of vaccination status. There was no statistically significant difference in Ct values based on the lineage regardless of the vaccination status. Recovery of infectious virus in cell culture was reduced in boosted patients compared to fully vaccinated without a booster and unvaccinated when infected with the Delta lineage. However, in patients with Omicron infections, recovery of infectious virus was not affected by vaccination. INTERPRETATION: Compared to Delta, Omicron was more likely to cause breakthrough infections of vaccinated individuals, yet admissions were less frequent. Admitted patients might develop severe disease comparable to Delta. Efforts for reducing Omicron transmission are required as, though the admission risk might be lower, the increased numbers of infections cause large numbers of hospitalizations. FUNDING: NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061, and The Modeling Infectious Diseases in Healthcare Network (MInD) under awards U01CK000589.

Large Scale SARS-CoV-2 Molecular Testing and Genomic Surveillance Reveal Prolonged Infections, Protracted RNA shedding, and Viral Reinfections.

Large-scale SARS-CoV-2 molecular testing coupled with whole genome sequencing in the diagnostic laboratories is instrumental for real-time genomic surveillance. The extensive genomic, laboratory, and clinical data provide a valuable resource for understanding cases of reinfection versus prolonged RNA shedding and protracted infections. In this study, data from a total of 22,292 clinical specimens, positive by SARS-CoV-2 molecular diagnosis at Johns Hopkins clinical virology laboratory between March 11th 2020 to September 23rd 2021, were used to identify patients with two or more positive results. A total of 3,650 samples collected from 1,529 patients who had between 2 and 20 positive results were identified in a time frame that extended up to 403 days from the first positive. Cycle threshold values (Ct) were available for 1,622 samples, the median of which was over 30 by 11 days after the first positive. Extended recovery of infectious virus on cell culture was notable for up to 70 days after the first positive in immunocompromised patients. Whole genome sequencing data generated as a part of our SARS-CoV-2 genomic surveillance was available for 1,027 samples from patients that had multiple positive tests. Positive samples collected more than 10 days after initial positive with high quality sequences (coverage >90% and mean depth >100), were more likely to be from unvaccinated, or immunosuppressed patients. Reinfections with viral variants of concern were found in 3 patients more than 130 days from prior infections with a different viral clade. In 75 patients that had 2 or more high quality sequences, the acquisition of more substitutions or deletions was associated with lack of vaccination and longer time between the recovered viruses. Our study highlights the value of integrating genomic, laboratory, and clinical data for understanding the biology of SARS-CoV-2 as well as for setting a precedent for future epidemics and pandemics.

A Quick Displacement of the SARS-CoV-2 variant Delta with Omicron: Unprecedented Spike in COVID-19 Cases Associated with Fewer Admissions and Comparable Upper Respiratory Viral Loads.

BACKGROUND: The increase in SARS-CoV-2 infections in December 2021 in the United States was driven primarily by the Omicron variant which largely displaced the Delta over a three week span. Outcomes from infection with the Omicron remain uncertain. We evaluate whether clinical outcomes and viral loads differ between Delta and Omicron infections during the period when both variants were co-circulating. METHODS: Remnant clinical specimens from patients that tested positive for SARS-CoV-2 after standard of care testing between the last week of November and the end of December 2021were used for whole viral genome sequencing. Cycle threshold values (Ct) for viral RNA, the presence of infectious virus, and levels of respiratory IgG were measured, and clinical outcomes were obtained. Differences in each measure were compared between variants stratified by vaccination status. RESULTS: The Omicron variant displaced the Delta during the study period and constituted 95% of the circulating lineages by the end of December 2021. Patients with Omicron infections (N= 1121) were more likely to be vaccinated compared to patients with Delta (N = 910), but were less likely to be admitted, require ICU level care, or succumb to infection regardless of vaccination status. There was no significant difference in Ct values based on the lineage regardless of the vaccination status. Recovery of infectious virus in cell culture was reduced in boosted patients compared to fully vaccinated without a booster and unvaccinated when infected with the Delta lineage. However, in patients with Omicron infections, recovery of infectious virus was not affected by vaccination. CONCLUSIONS: Omicron infections of vaccinated individuals are expected, yet admissions are less frequent. Admitted patients might develop severe disease comparable to Delta. Efforts for reducing the Omicron transmission are required as even though the admission risk is lower, the numbers of infections continue to be high. RESEARCH IN CONTEXT EVIDENCE BEFORE THIS STUDY: The unprecedented increase in COVID-19 cases in the month of December 2021, associated with the displacement of the Delta variant with the Omicron, triggered a lot of concerns. An understanding of the disease severity associated with infections with Omicron is essential as well as the virological determinants that contributed to its widespread predominance. We searched PubMed for articles published up to January 23, 2022, using the search terms ("Omicron") AND ("Disease severity") as well as ("Omicron") AND ("Viral load") And/ or ("Cell culture"). Our search yielded 3 main studies that directly assessed the omicron's clinical severity in South Africa, its infectious viral load compared to Delta, and the dynamics of viral RNA shedding. In South Africa, compared to Delta, Omicron infected patients showed a significant reduction in severe disease. In this study, Omicron and non-Omicron variants were characterized based on S gene target failure using the TaqPath COVID-19 PCR (Thermo Fisher Scientific). In the study from Switzerland that assessed the infectious viral load in Omicron versus Delta, the authors analyzed only 18 Omicron samples that were all from vaccinated individuals to show that compared to Delta, Omicron had equivalent infectious viral titers. The third study that assessed the Omicron viral dynamics showed that the peak viral RNA in Omicron infections is lower than Delta. No published studies assessed the clinical discrepancies of Omicron and Delta infected patients from the US, nor comprehensively assessed, by viral load and cell culture studies, the characteristics of both variants stratified by vaccination status. ADDED VALUE OF THIS STUDY: To the best of our knowledge, this is the only study to date to compare the clinical characteristics and outcomes after infection with the Omicron variant compared to Delta in the US using variants characterized by whole genome sequencing and a selective time frame when both variant co-circulated. It is also the first study to stratify the analysis based on the vaccination status and to compare fully vaccinated patients who didn't receive a booster vaccination to patients who received a booster vaccination. In addition, we provide a unique viral RNA and infectious virus load analyses to compare Delta and Omicron samples from unvaccinated, fully vaccinated, and patients with booster vaccination. IMPLICATIONS OF ALL THE AVAILABLE EVIDENCE: Omicron associated with a significant increase in infections in fully and booster vaccinated individuals but with less admissions and ICU level care. Admitted patients showed similar requirements for supplemental oxygen and ICU level care when compared to Delta admitted patients. Viral loads were similar in samples from Omicron and Delta infected patients regardless of the vaccination status. The recovery of infectious virus on cell culture was reduced in samples from patients infected with Delta who received a booster dose, but this was not the case with Omicron. The recovery of infectious virus was equivalent in Omicron infected unvaccinated, fully vaccinated, and samples from patients who received booster vaccination. FUNDING: NIH/NIAID Center of Excellence in Influenza Research and Surveillance contract HHS N2772201400007C, Johns Hopkins University, Maryland department of health, Centers for Disease Control and Prevention contract 75D30121C11061.

Impact of SARS-CoV-2 variants on inpatient clinical outcome.

BACKGROUND: Prior observation has shown differences in COVID-19 hospitalization rates between SARS-CoV-2 variants, but limited information describes differences in hospitalization outcomes. METHODS: Patients admitted to 5 hospitals with COVID-19 were included if they had hypoxia, tachypnea, tachycardia, or fever, and data to describe SARS-CoV-2 variant, either from whole genome sequencing, or inference when local surveillance showed ≥95% dominance of a single variant. The average effect of SARS-CoV-2 variant on 14-day risk of severe disease, defined by need for advanced respiratory support, or death was evaluated using models weighted on propensity scores derived from baseline clinical features. RESULTS: Severe disease or death within 14 days occurred for 950 of 3,365 (28%) unvaccinated patients and 178 of 808 (22%) patients with history of vaccination or prior COVID-19. Among unvaccinated patients, the relative risk of 14-day severe disease or death for Delta variant compared to ancestral lineages was 1.34 (95% confidence interval [CI] 1.13-1.55). Compared to Delta variant, this risk for Omicron patients was 0.78 (95% CI 0.62-0.97) and compared to ancestral lineages was 1.04 (95% CI 0.84-1.24). Among Omicron and Delta infections, patients with history of vaccination or prior COVID-19 had one-half the 14-day risk of severe disease or death (adjusted hazard ratio 0.46, IQR 0.34-0.62) but no significant outcome difference between Delta and Omicron infections. CONCLUSIONS: Although the risk of severe disease or death for unvaccinated patients with Omicron was lower than Delta, it was similar to ancestral lineages. Severe outcomes were less common in vaccinated patients, but there was no difference between Delta and Omicron infections.

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.