COVID-19 vaccinated children, adolescents, and young adults with acute lymphoblastic leukemia show spike reactive antibodies and multifunctional T-cells.

Little is known about the efficacy of COVID-19 vaccines during acute lymphoblastic leukemia therapy (ALL); data for COVID-19 vaccine immune responses in pediatric leukemia remain sparse. We conducted a single center study of patients aged 5-25 years undergoing ALL chemotherapy who received COVID-19 vaccination. Twenty-one patients were enrolled; efficacy was evaluable in 20. Twenty were vaccinated while receiving chemotherapy. Twenty received the BNT162b2 mRNA vaccine. Spike reactive antibodies (S-IgG) and/or T-cells (SRT) were detected in 16 of 20 (80%) vaccinated patients; 13 (65%) and 9 (45%) were positive for S-IgG and SRT, respectively. Six (30%) showed both spike reactive B and T-cell responses. Eleven of the 13 with S-IgG positivity were negative for anti-Nucleocapsid IgG, an antibody profile consistent with a vaccine induced immune response. All 13S-IgG+ patients showed neutralizing antibodies. SRT included CD4+ (7) and CD8+ (6) T-cells; both CD4+ and CD8+ SRT were seen in 4. SRT were multifunctional (producing multiple cytokines) in most patients (8 of 9); 4 showed SRT with triple cytokine and B-cell co-stimulatory responses, indicating a multimodal adaptive immune response. Immune responses were seen among patients vaccinated in the settings of lymphopenia (6 of 12) intensive chemotherapy (3 of 4), and Peg allergy (6 of 8). Sequencing revealed public CD4+ and CD8+ TCR sequences reactive to epitopes across the spike protein. In conclusion, COVID-19 vaccination induced B and/or T-cell responses in a majority of children and young adults undergoing ALL chemotherapy.

Rapid antimicrobial resistance detection methods for bloodstream infection in solid organ transplantation: Proposed clinical guidance, unmet needs, and future directions.

Recent advances in antimicrobial resistance detection have spurred the development of multiple assays that can accurately detect the presence of bacterial resistance from positive blood cultures, resulting in faster institution of effective antimicrobial therapy. Despite these advances, there are limited data regarding the use of these assays in solid organ transplant (SOT) recipients and there is little guidance on how to select, implement, and interpret them in clinical practice. We describe a practical approach to the implementation and interpretation of these assays in SOT recipients using the best available data and expert opinion. These findings were part of a consensus conference sponsored by the American Society of Transplantation held on December 7, 2021 and represent the collaboration between experts in transplant infectious diseases, pharmacy, antimicrobial and diagnostic stewardship, and clinical microbiology. Areas of unmet need and recommendations for future investigation are also presented.

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.

Impact of FilmArray™ Respiratory Panel testing on the clinical management of pediatric bone marrow transplant patients.

Viral respiratory tract infections cause significant morbidity in bone marrow transplant (BMT) patients. Speed and sensitivity of the FilmArray™ Respiratory Panel (FA-RP) can improve care but may prompt inappropriate testing. Studies describing FA-RP use in pediatric BMT patients are limited; we investigated FA-RP use, results, and clinical management to evaluate clinical significance of testing in pediatric BMT patients. Retrospective analysis of 671 respiratory specimens from 204 unique BMT patients between 01/01/2016 and 01/01/2019 was performed. Age, underlying diagnoses, FA-RP result, reason for FA-RP, and symptoms were abstracted. FA-RP impact on antimicrobial management, scheduled procedures, infection control measures, and hospital admission/discharge were investigated. Impacts of repeat testing were evaluated. Two hundred sixty-nine out of 671 specimens (40%) tested positive; human rhinovirus/enterovirus (hRV/hEV) was the most common (161/269, 60%). The primary reason for FA-RP was URI symptoms (402/671, 60%) with 54% testing positive. One hundred twenty-two out of 671 (18.2%) specimens were from asymptomatic patients; 14 (11.4%) tested positive. FA-RP informed antiviral initiation in 7/19 (36.8%), 7/8 (87.5%), and 5/30 (16.7%) of RSV, influenza, and human parainfluenza cases, respectively. In 11 cases, FA-RP informed azithromycin and ceftriaxone initiation, continuation, or discontinuation. BMT was delayed for three positives (two RSV, one hRV/hEV). In 22 instances, negative FA-RP cleared patients for BMT. In 70% of cases, repeats offered no new clinical information; all negative-to-positive cases had new or worsening respiratory symptoms. FA-RP was ordered on symptomatic and asymptomatic patients, provided rapid diagnosis in > 50% of symptomatic patients, and informed infection control measures for all inpatients and antiviral initiation in > 80% of influenza cases.

Monkeypox: Clinical Considerations, Epidemiology, and Laboratory Diagnostics.

Monkeypox virus (MPXV) has garnered recent attention as outbreaks are continually reported outside historic regions of endemicity in Africa. Consequently, MPXV is becoming routinely included in the differential diagnosis of rash illnesses, requiring clinicians and laboratorians alike to quickly adapt to a new public health emergency. This review discusses the epidemiology, clinical presentation, and laboratory testing of MPXV in the context of recent outbreaks.

A multicenter evaluation of viral bloodstream detections in children presenting to the Emergency Department with suspected systemic infection.

BACKGROUND: Fever is a common symptom in children presenting to the Emergency Department (ED). We aimed to describe the epidemiology of systemic viral infections and their predictive values for excluding serious bacterial infections (SBIs), including bacteremia, meningitis and urinary tract infections (UTIs) in children presenting to the ED with suspected systemic infections. METHODS: We enrolled children who presented to the ED with suspected systemic infections who had blood cultures obtained at seven healthcare facilities. Whole blood specimens were analyzed by an experimental multiplexed PCR test for 7 viruses. Demographic and laboratory results were abstracted. RESULTS: Of the 1114 subjects enrolled, 245 viruses were detected in 224 (20.1%) subjects. Bacteremia, meningitis and UTI frequency in viral bloodstream-positive patients was 1.3, 0 and 10.1% compared to 2.9, 1.3 and 9.7% in viral bloodstream-negative patients respectively. Although viral bloodstream detections had a high negative predictive value for bacteremia or meningitis (NPV = 98.7%), the frequency of UTIs among these subjects remained appreciable (9/89, 10.1%) (NPV = 89.9%). Screening urinalyses were positive for leukocyte esterase in 8/9 (88.9%) of these subjects, improving the ability to distinguish UTI. CONCLUSIONS: Viral bloodstream detections were common in children presenting to the ED with suspected systemic infections. Although overall frequencies of SBIs among subjects with and without viral bloodstream detections did not differ significantly, combining whole blood viral testing with urinalysis provided high NPV for excluding SBI.

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.

Why Can't We Just Use PCR? The Role of Genotypic versus Phenotypic Testing for Antimicrobial Resistance Testing.

There is a need for phenotypic susceptibility testing that is expeditious and that can be performed directly from clinical specimens. While rapid pathogen identification is important, it is the susceptibility result that is essential for antimicrobial optimization. The options for rapid susceptibility testing are limited, with the majority of commercial tests available offering genotypic resistance detection only. In this article, a laboratorian and a clinician discuss the benefits and limitations of genotypic and phenotypic susceptibility testing and provide examples of how results should be interpreted to maximize the clinical utility.

Performance of the CLSI Carba NP and the Rosco Carb Screen Assays Using North American Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa Isolates.

This study compared the performance of the Carba NP assay, published by the Clinical and Laboratory Standards Institute, and the Rosco Rapid Carb Screen kit. Carba NP had superior sensitivity, but both assays required an increased inoculum to detect carbapenemase production in isolates with blaNDM, blaIMP, and blaOXA-48.

SARS-CoV-2 Variants from Long-Term, Persistently Infected Immunocompromised Patients Have Altered Syncytia Formation, Temperature-Dependent Replication, and Serum Neutralizing Antibody Escape.

SARS-CoV-2 infection of immunocompromised individuals often leads to prolonged detection of viral RNA and infectious virus in nasal specimens, presumably due to the lack of induction of an appropriate adaptive immune response. Mutations identified in virus sequences obtained from persistently infected patients bear signatures of immune evasion and have some overlap with sequences present in variants of concern. We characterized virus isolates obtained greater than 100 days after the initial COVID-19 diagnosis from two COVID-19 patients undergoing immunosuppressive cancer therapy, wand compared them to an isolate from the start of the infection. Isolates from an individual who never mounted an antibody response specific to SARS-CoV-2 despite the administration of convalescent plasma showed slight reductions in plaque size and some showed temperature-dependent replication attenuation on human nasal epithelial cell culture compared to the virus that initiated infection. An isolate from another patient-who did mount a SARS-CoV-2 IgM response-showed temperature-dependent changes in plaque size as well as increased syncytia formation and escape from serum-neutralizing antibodies. Our results indicate that not all virus isolates from immunocompromised COVID-19 patients display clear signs of phenotypic change, but increased attention should be paid to monitoring virus evolution in this patient population.

Detection of mecA-mediated methicillin resistance and evaluation of disk-diffusion antimicrobial susceptibility characteristics of Staphylococcus saprophyticus isolates from geographically diverse locations: Staphylococcus saprophyticus antibiotic susceptibility testing.

Staphylococcus saprophyticus, a common uropathogen, is usually susceptible to urine-concentrating antimicrobials, so routine AST is not recommended by CLSI. Our study evaluated the antimicrobial resistance profiles of 277 S. saprophyticus isolates from North America and a globally diverse cohort. Notably, 24% (67/277) of our isolates come from non-urinary sources. AST was performed against 12 antimicrobials using standard disk diffusion, PCR for mecA and mecC, PBP2a production assays, and cefinase. 5% (13/277) of isolates were mecA positive and cefinase positive, 63% (176/277) were mecA negative but cefinase positive, 4% (11/277) were mecA positive but cefinase negative, and 28% (77/277) were mecA and cefinase negative. All (277/277) isolates were susceptible to delafloxacin, ciprofloxacin, rifampin, linezolid, and nitrofurantoin and 95% (262/277) were susceptible to trimethoprim-sulfamethoxazole. Our results showed that regardless of using CLSI or EUCAST breakpoints oxacillin had low categorical agreement for mecA presence, making it unsuitable for surrogate testing, while cefoxitin disk diffusion had high very major error rate. If possible, PBP2a or mecA testing is recommended for guiding therapy for non-urinary infections. Our work supports CLSI guidelines on routine susceptibility to urinary tract antibiotics.

Etiologies of bloody diarrhea in children presenting with acute gastroenteritis to US emergency departments.

Among 111 children presenting with bloody diarrhea in a multicenter study of molecular testing in US emergency departments, we found viral pathogens in 18%, bacteria in 48%, protozoa in 2%, and no pathogens detected in 38%.

Considerations for SARS-CoV-2 Genomic Surveillance: A Joint Consensus Recommendation of the Association for Molecular Pathology and Association of Public Health Laboratories.

Next-generation sequencing (NGS) has applications in research, epidemiology, oncology, and infectious disease diagnostics. Wide variability exists in NGS wet laboratory techniques and dry laboratory analytical considerations. Thus, many questions remain unanswered when NGS methods are implemented in laboratories for infectious disease testing. While this review is not intended to answer all questions, the most pressing questions from a public health and clinical hospital-based laboratory perspective will be addressed. The authors of this review are laboratory professionals who perform and interpret SARS-CoV-2 NGS results. Considerations for pre-analytical, analytical, and post-analytical NGS will be explored. This review highlights challenges for molecular laboratory professionals considering adopting or expanding NGS methods.

Exploring the Utility of Multiplex Infectious Disease Panel Testing for Diagnosis of Infection in Different Body Sites: A Joint Report of the Association for Molecular Pathology, American Society for Microbiology, Infectious Diseases Society of America, and Pan American Society for Clinical Virology.

The use of clinical molecular diagnostic methods for detecting microbial pathogens continues to expand and, in some cases, supplant conventional identification methods in various scenarios. Analytical and clinical benefits of multiplex molecular panels for the detection of respiratory pathogens have been demonstrated in various studies. The use of these panels in managing different patient populations has been incorporated into clinical guidance documents. The Association for Molecular Pathology's Infectious Diseases Multiplex Working Group conducted a review of the current benefits and challenges to using multiplex PCR for the detection of pathogens from gastrointestinal tract, central nervous system, lower respiratory tract, and joint specimens. The Working Group also discusses future directions and novel approaches to detection of pathogens in alternate specimen types, and outlines challenges associated with implementation of these multiplex PCR panels.

Clinical Impact of Multiplex Molecular Diagnostic Testing in Children with Acute Gastroenteritis Presenting to An Emergency Department: A Multicenter Prospective Study.

Background: Multiplex molecular diagnostic panels have greatly enhanced detection of gastrointestinal pathogens. However, data on the impact of these tests on clinical and patient-centered outcomes are limited. Methods: We conducted a prospective, multicenter, stepped-wedge trial to determine the impact of multiplex molecular testing at five academic children's hospitals in children presenting to the ED with acute gastroenteritis. Caregivers were interviewed on enrollment and again 7-10 days after enrollment to determine symptoms, risk factors, subsequent medical visits, and impact on family members. During the pre-intervention period, diagnostic testing was performed at the discretion of clinicians. During the intervention period, multiplex molecular testing was performed on all children with results available to clinicians. Primary outcome was return visits to a health care provider within 10 days of enrollment. Results: Potential pathogens were identified by clinician ordered tests in 19/571 (3.3%) in the pre-intervention period compared to 434/586 (74%) in the intervention period; clinically relevant pathogens were detected in 2.1% and 15% respectively. In the multivariate model adjusting for potential confounders, the intervention was associated with a 21% reduction in the odds of any return visit (OR 0.79; 95% CI 0.70-0.90). Appropriate treatment was prescribed in 11.3% compared to 19.6% during the intervention period(P=0.22). Conclusions: Routine molecular multiplex testing for all children presenting to the ED with AGE detected more clinically relevant pathogens and led to a 21% decrease in return visits. Additional research is needed to define patients most likely to benefit from testing.

Distinguishing cytomegalovirus meningoencephalitis from other viral central nervous system infections.

BACKGROUND: Hallmarks of cytomegalovirus (CMV) meningoencephalitis include fever, altered mental status, or meningismus with pleocytosis, elevated protein and hypoglycorrhachia on cerebrospinal fluid (CSF) analysis. Magnetic resonance imaging may show ventriculitis, ependymitis or periventricular enhancement. Studies are limited comparing clinical and laboratory characteristics to other viral etiologies. OBJECTIVES: This multi-center, retrospective cohort analysis reviewed patients with CMV meningitis or encephalitis and compared clinical features, laboratory findings and outcomes to the most common viral causes of meningoencephalitis. STUDY DESIGN: Patients with encephalitis or aseptic meningitis and detectable genetic material by polymerase chain reaction were identified. Clinical characteristics, laboratory findings and neuroimaging were collected from the electronic medical record. Data analysis was performed comparing CMV to other viral etiologies. RESULTS: 485 patients were evaluated and included cases of CMV (n = 36) which were compared with herpes simplex virus (n = 114), enterovirus (n = 207), varicella zoster virus (n = 41) and West Nile virus (n = 81). Human immunodeficiency virus (HIV) infection was seen more frequently in CMV infection compared with all other viral etiologies. Clinical presentations and CSF findings of other viral etiologies differ compared with CMV. Hypoglycorrhacia occurred more often with CMV compared with other viral pathogens. Outcomes were significantly worse compared with enterovirus, herpes simplex virus and varicella zoster virus but not West Nile virus. CONCLUSIONS: CMV meningoencephalitis occurs most often in patients with HIV and encephalitis occurs more frequently than meningitis. Clinical and laboratory findings differ compared with other viral etiologies and can support consideration of CMV in the differential diagnosis of patients with meningoencephalitis.

Rapidly emerging SARS-CoV-2 B.1.1.7 sub-lineage in the United States of America with spike protein D178H and membrane protein V70L mutations.

The SARS-CoV-2 B.1.1.7 lineage is highly infectious and as of April 2021 accounted for 92% of COVID-19 cases in Europe and 59% of COVID-19 cases in the U.S. It is defined by the N501Y mutation in the receptor-binding domain (RBD) of the Spike (S) protein, and a few other mutations. These include two mutations in the N terminal domain (NTD) of the S protein, HV69-70del and Y144del (also known as Y145del due to the presence of tyrosine at both positions). We recently identified several emerging SARS-CoV-2 variants of concerns, characterized by Membrane (M) protein mutations, including I82T and V70L. We now identify a sub-lineage of B.1.1.7 that emerged through sequential acquisitions of M:V70L in November 2020 followed by a novel S:D178H mutation first observed in early February 2021. The percentage of B.1.1.7 isolates in the US that belong to this sub-lineage increased from 0.15% in February 2021 to 1.8% in April 2021. To date, this sub-lineage appears to be U.S.-specific with reported cases in 31 states, including Hawaii. As of April 2021, it constituted 36.8% of all B.1.1.7 isolates in Washington. Phylogenetic analysis and transmission inference with Nextstrain suggest this sub-lineage likely originated in either California or Washington. Structural analysis revealed that the S:D178H mutation is in the NTD of the S protein and close to two other signature mutations of B.1.1.7, HV69-70del and Y144del. It is surface exposed and may alter NTD tertiary configuration or accessibility, and thus has the potential to affect neutralization by NTD directed antibodies.

Emerging variants of concern in SARS-CoV-2 membrane protein: a highly conserved target with potential pathological and therapeutic implications.

Mutations in the SARS-CoV-2 Membrane (M) gene are relatively uncommon. The M gene encodes the most abundant viral structural protein, and is implicated in multiple viral functions, including initial attachment to the host cell via heparin sulphate proteoglycan, viral protein assembly in conjunction with the N and E genes, and enhanced glucose transport. We have identified a recent spike in the frequency of reported SARS-CoV-2 genomes carrying M gene mutations. This is associated with emergence of a new sub-B.1 clade, B.1.I82T, defined by the previously unreported M:I82T mutation within TM3, the third of three membrane spanning helices implicated in glucose transport. The frequency of this mutation increased in the USA from 0.014% in October 2020 to 1.62% in February 2021, a 116-fold change. While constituting 0.7% of the isolates overall, M:I82T sub-B.1 lineage accounted for 14.4% of B.1 lineage isolates in February 2021, similar to the rapid initial increase previously seen with the B.1.1.7 and B.1.429 lineages, which quickly became the dominant lineages in Europe and California over a period of several months. A similar increase in incidence was also noted in another related mutation, V70L, also within the TM2 transmembrane helix. These M mutations are associated with younger patient age (4.6 to 6.3 years). The rapid emergence of this B.1.I82T clade, recently named Pangolin B.1.575 lineage, suggests that this M gene mutation is more biologically fit, perhaps related to glucose uptake during viral replication, and should be included in ongoing genomic surveillance efforts and warrants further evaluation for potentially increased pathogenic and therapeutic implications.

Early pandemic molecular diversity of SARS-CoV-2 in children.

Background: In the US, community circulation of the SARS-CoV-2 virus likely began in February 2020 after mostly travel-related cases. Children's Hospital of Philadelphia began testing on 3/9/2020 for pediatric and adult patients, and for all admitted patients on 4/1/2020, allowing an early glimpse into the local molecular epidemiology of the virus. Methods: We obtained 169 SARS-CoV-2 samples (83 from patients <21 years old) from March through May and produced whole genome sequences. We used genotyping tools to track variants over time and to test for possible genotype associated clinical presentations and outcomes in children. Results: Our analysis uncovered 13 major lineages that changed in relative abundance as cases peaked in mid-April in Philadelphia. We detected at least 6 introductions of distinct viral variants into the population. As a group, children had more diverse virus genotypes than the adults tested. No strong differences in clinical variables were associated with genotypes. Conclusions: Whole genome analysis revealed unexpected diversity, and distinct circulating viral variants within the initial peak of cases in Philadelphia. Most introductions appeared to be local from nearby states. Although limited by sample size, we found no evidence that different genotypes had different clinical impacts in children in this study.