Researching COVID to enhance recovery (RECOVER) pediatric study protocol: Rationale, objectives and design.

IMPORTANCE: The prevalence, pathophysiology, and long-term outcomes of COVID-19 (post-acute sequelae of SARS-CoV-2 [PASC] or "Long COVID") in children and young adults remain unknown. Studies must address the urgent need to define PASC, its mechanisms, and potential treatment targets in children and young adults. OBSERVATIONS: We describe the protocol for the Pediatric Observational Cohort Study of the NIH's REsearching COVID to Enhance Recovery (RECOVER) Initiative. RECOVER-Pediatrics is an observational meta-cohort study of caregiver-child pairs (birth through 17 years) and young adults (18 through 25 years), recruited from more than 100 sites across the US. This report focuses on two of four cohorts that comprise RECOVER-Pediatrics: 1) a de novo RECOVER prospective cohort of children and young adults with and without previous or current infection; and 2) an extant cohort derived from the Adolescent Brain Cognitive Development (ABCD) study (n = 10,000). The de novo cohort incorporates three tiers of data collection: 1) remote baseline assessments (Tier 1, n = 6000); 2) longitudinal follow-up for up to 4 years (Tier 2, n = 6000); and 3) a subset of participants, primarily the most severely affected by PASC, who will undergo deep phenotyping to explore PASC pathophysiology (Tier 3, n = 600). Youth enrolled in the ABCD study participate in Tier 1. The pediatric protocol was developed as a collaborative partnership of investigators, patients, researchers, clinicians, community partners, and federal partners, intentionally promoting inclusivity and diversity. The protocol is adaptive to facilitate responses to emerging science. CONCLUSIONS AND RELEVANCE: RECOVER-Pediatrics seeks to characterize the clinical course, underlying mechanisms, and long-term effects of PASC from birth through 25 years old. RECOVER-Pediatrics is designed to elucidate the epidemiology, four-year clinical course, and sociodemographic correlates of pediatric PASC. The data and biosamples will allow examination of mechanistic hypotheses and biomarkers, thus providing insights into potential therapeutic interventions. CLINICAL TRIALS.GOV IDENTIFIER: Clinical Trial Registration: http://www.clinicaltrials.gov. Unique identifier: NCT05172011.

Early Estimate of Nirsevimab Effectiveness for Prevention of Respiratory Syncytial Virus-Associated Hospitalization Among Infants Entering Their First Respiratory Syncytial Virus Season - New Vaccine Surveillance Network, October 2023-February 2024.

Respiratory syncytial virus (RSV) is the leading cause of hospitalization among infants in the United States. In August 2023, CDC's Advisory Committee on Immunization Practices recommended nirsevimab, a long-acting monoclonal antibody, for infants aged <8 months to protect against RSV-associated lower respiratory tract infection during their first RSV season and for children aged 8-19 months at increased risk for severe RSV disease. In phase 3 clinical trials, nirsevimab efficacy against RSV-associated lower respiratory tract infection with hospitalization was 81% (95% CI = 62%-90%) through 150 days after receipt; post-introduction effectiveness has not been assessed in the United States. In this analysis, the New Vaccine Surveillance Network evaluated nirsevimab effectiveness against RSV-associated hospitalization among infants in their first RSV season during October 1, 2023-February 29, 2024. Among 699 infants hospitalized with acute respiratory illness, 59 (8%) received nirsevimab ≥7 days before symptom onset. Nirsevimab effectiveness was 90% (95% CI = 75%-96%) against RSV-associated hospitalization with a median time from receipt to symptom onset of 45 days (IQR = 19-76 days). The number of infants who received nirsevimab was too low to stratify by duration from receipt; however, nirsevimab effectiveness is expected to decrease with increasing time after receipt because of antibody decay. Although nirsevimab uptake and the interval from receipt of nirsevimab were limited in this analysis, this early estimate supports the current nirsevimab recommendation for the prevention of severe RSV disease in infants. Infants should be protected by maternal RSV vaccination or infant receipt of nirsevimab.

Prevalence and clinical presentation of EV-D68 infections in Kansas City children during the 2022 season.

Seasonal EV-D68 infections can strain medical care resources due to increased pediatric hospitalizations for respiratory illness. In this study, we examine Kansas City's 2022 EV-D68 season. Rhinovirus/enterovirus (RV/EV) positive respiratory specimens from standard of care testing were salvaged and tested by EV-D68 specific PCR. Of the 1412 respiratory specimens tested from July 1 to September 15, 2022, 346 (23%) were positive for RV/EV and EV-D68 was detected in 134/319 (42%) salvaged RV/EV positive specimens. The median age of children with EV-D68 infections was 35.2 months (IQR 16.1, 67.3), which was older than children with non-EV-D68 RV/EV infections (16 months, IQR 5, 47.8), but younger than children infected during the 2014 EV-D68 outbreak. EV-D68 infection was more likely to cause severe disease in children with asthma compared to those without asthma. Real-time EV-D68 monitoring for outbreaks could potentially improve resource utilization by hospitals and help prepare for surges of respiratory disease.

Researching COVID to enhance recovery (RECOVER) pediatric study protocol: Rationale, objectives and design.

Importance: The prevalence, pathophysiology, and long-term outcomes of COVID-19 (post-acute sequelae of SARS-CoV-2 [PASC] or "Long COVID") in children and young adults remain unknown. Studies must address the urgent need to define PASC, its mechanisms, and potential treatment targets in children and young adults. Observations: We describe the protocol for the Pediatric Observational Cohort Study of the NIH's RE searching COV ID to E nhance R ecovery (RECOVER) Initiative. RECOVER-Pediatrics is an observational meta-cohort study of caregiver-child pairs (birth through 17 years) and young adults (18 through 25 years), recruited from more than 100 sites across the US. This report focuses on two of five cohorts that comprise RECOVER-Pediatrics: 1) a de novo RECOVER prospective cohort of children and young adults with and without previous or current infection; and 2) an extant cohort derived from the Adolescent Brain Cognitive Development (ABCD) study ( n =10,000). The de novo cohort incorporates three tiers of data collection: 1) remote baseline assessments (Tier 1, n=6000); 2) longitudinal follow-up for up to 4 years (Tier 2, n=6000); and 3) a subset of participants, primarily the most severely affected by PASC, who will undergo deep phenotyping to explore PASC pathophysiology (Tier 3, n=600). Youth enrolled in the ABCD study participate in Tier 1. The pediatric protocol was developed as a collaborative partnership of investigators, patients, researchers, clinicians, community partners, and federal partners, intentionally promoting inclusivity and diversity. The protocol is adaptive to facilitate responses to emerging science. Conclusions and Relevance: RECOVER-Pediatrics seeks to characterize the clinical course, underlying mechanisms, and long-term effects of PASC from birth through 25 years old. RECOVER-Pediatrics is designed to elucidate the epidemiology, four-year clinical course, and sociodemographic correlates of pediatric PASC. The data and biosamples will allow examination of mechanistic hypotheses and biomarkers, thus providing insights into potential therapeutic interventions. Clinical Trialsgov Identifier: Clinical Trial Registration: http://www.clinicaltrials.gov . Unique identifier: NCT05172011.

Comparative analysis of three multiplex platforms for the detection of respiratory viral pathogens.

BACKGROUND: Acute viral respiratory infections are a major health burden in children worldwide. In recent years, rapid and sensitive multiplex nucleic acid amplification tests (NAATs) have replaced conventional methods for routine virus detection in the clinical laboratory. OBJECTIVE/STUDY DESIGN: We compared BioFire® FilmArray® Respiratory Panel (FilmArray V1.7), Luminex NxTag® Respiratory Pathogen Panel (NxTag RPP) and Applied Biosystems TaqMan Array Card (TAC) for the detection of eight viruses in pediatric respiratory specimens. Results from the three platforms were analyzed with a single-plex real-time RT-PCR (rRT-PCR) assay for each virus. RESULTS: Of the 170/210 single-plex virus-positive samples, FilmArray detected a virus in 166 (97.6%), TAC in 163 (95.8%) and NxTag RPP in 160 (94.1%) samples. The Positive Percent Agreement (PPA) of FilmArray, NxTag RPP and TAC was highest for influenza B (100%, 100% and 95.2% respectively) and lowest for seasonal coronaviruses on both FilmArray (90.2%) and NxTag RPP (81.8%), and for parainfluenza viruses 1- 4 on TAC (84%). The Negative Percent Agreement (NPA) was lowest for rhinovirus/enterovirus (92.9%, 96.7% and 97.3%) on FilmArray, NxTag RPP and TAC respectively. NPA for all three platforms was highest (100%) for both parainfluenza viruses 1- 4 and influenza A and B, and 100% for human metapneumovirus with TAC as well. CONCLUSION: All three multiplex platforms displayed high overall agreement (>90%) and high NPA (>90%), while PPA was pathogen dependent and varied among platforms; high PPA (>90%) was observed for FilmArray for all eight viruses, TAC for six viruses and NxTag RPP for 4 viruses.

The Impact of Prior Infection and Age on Antibody Persistence After Severe Acute Respiratory Syndrome Coronavirus 2 Messenger RNA Vaccine.

Determining the duration of immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines is critical for informing the timing of booster immunization. Many genetic and environmental factors could influence both the magnitude and persistence of the antibody response. Here, we showed that SARS-CoV-2 infection before vaccination and age affected the decay of antibody responses to the SARS-CoV-2 messenger RNA vaccine.

Comparison of diagnostic performance of five molecular assays for detection of SARS-CoV-2.

We compared the performance of the Abbott Real Time SARS-CoV-2 assay (Abbott assay), Aptima™ SARS-CoV-2 assay (Aptima assay), BGI Real-Time SARS-CoV-2 assay (BGI assay), Lyra® SARS-CoV-2 assay (Lyra assay), and DiaSorin Simplexa™ COVID assay for SARS-CoV-2 detection. Residual nasopharyngeal samples (n = 201) submitted for routine SARS-CoV-2 testing by Simplexa assay during June-July 2020 and January 2021 were salvaged. Aliquots were tested on other assays and compared against the CDC 2019-nCoV Real-Time RT-PCR assay. Viral load in positive samples was determined by droplet digital PCR. Among 201 samples, 99 were positive and 102 were negative by the CDC assay. The Aptima and Abbott assays exhibited the highest positive percent agreement (PPA) at 98.9% while the BGI assay demonstrated the lowest PPA of 89.9% with 10 missed detections. Negative percent agreement for all 5 platforms was comparable, ranging from 96.1% to 100%. The performance of all five assays was comparable.

Immune cell residency in the nasal mucosa may partially explain respiratory disease severity across the age range.

Previous studies focusing on the age disparity in COVID-19 severity have suggested that younger individuals mount a more robust innate immune response in the nasal mucosa after infection with SARS-CoV-2. However, it is unclear if this reflects increased immune activation or increased immune residence in the nasal mucosa. We hypothesized that immune residency in the nasal mucosa of healthy individuals may differ across the age range. We applied single-cell RNA-sequencing and measured the cellular composition and transcriptional profile of the nasal mucosa in 35 SARS-CoV-2 negative children and adults, ranging in age from 4 months to 65 years. We analyzed in total of ~ 30,000 immune and epithelial cells and found that age and immune cell proportion in the nasal mucosa are inversely correlated, with little evidence for structural changes in the transcriptional state of a given cell type across the age range. Orthogonal validation by epigenome sequencing indicate that it is especially cells of the innate immune system that underlie the age-association. Additionally, we characterize the predominate immune cell type in the nasal mucosa: a resident T cell like population with potent antiviral properties. These results demonstrate fundamental changes in the immune cell makeup of the uninfected nasal mucosa over the lifespan. The resource we generate here is an asset for future studies focusing on respiratory infection and immunization strategies.

Diagnostic Yield of Saliva for SARS-CoV-2 Molecular Testing in Children.

Pediatric saliva specimen demonstrated high sensitivity (93%) and specificity (96.2%) compared to paired nasopharyngeal swabs (NPS) by Aptima SARS-CoV-2 Assay (Aptima). Viral loads were comparable in both specimen types. Saliva is a safe, noninvasive, and acceptable alternative specimen for SARS-CoV-2 detection in children.

Humoral immune responses during SARS-CoV-2 mRNA vaccine administration in seropositive and seronegative individuals.

BACKGROUND: The global pandemic of coronavirus disease 2019 (COVID-19) is caused by infection with the SARS-CoV-2 virus. Currently, there are three approved vaccines against SARS-CoV-2 in the USA, including two based on messenger RNA (mRNA) technology that has demonstrated high vaccine efficacy. We sought to characterize humoral immune responses, at high resolution, during immunization with the BNT162b2 (Pfizer-BioNTech) vaccine in individuals with or without prior history of natural SARS-CoV-2 infection. METHODS: We determined antibody responses after each dose of the BNT162b2 SARS-CoV-2 vaccine in individuals who had no prior history of SARS-CoV-2 infection (seronegative) and individuals that had previous viral infection 30-60 days prior to first vaccination (seropositive). To do this, we used both an antibody isotype-specific multiplexed bead-based binding assays targeting multiple SARS-CoV-2 viral protein antigens and an assay that identified potential SARS-CoV-2 neutralizing antibody levels. Moreover, we mapped antibody epitope specificity after immunization using SARS-CoV-2 spike protein peptide arrays. RESULTS: Antibody levels were significantly higher after a single dose in seropositive individuals compared to seronegative individuals and were comparable to levels observed in seronegative individuals after two doses. While IgG was boosted by vaccination for both seronegative and seropositive individuals, only seronegative individuals had increased IgA or IgM antibody titers after primary immunization. We identified immunodominant peptides targeted on both SARS-CoV-2 spike S1 and S2 subunits after vaccination. CONCLUSION: These findings demonstrated the antibody responses to SARS-CoV-2 immunization in seropositive and seronegative individuals and provide support for the concept of using prior infection history as a guide for the consideration of future vaccination regimens. Moreover, we identified key epitopes on the SARS-CoV-2 spike protein that are targeted by antibodies after vaccination that could guide future vaccine and immune correlate development.

Comparative in vitro effectiveness of ceftolozane/tazobactam against pediatric gram-negative drug-resistant isolates.

Ceftolozane/tazobactam (C/T), a cephalosporin/beta-lactamase inhibitor combination, was evaluated in vitro vs. 10 comparators against 299 pediatric extended-spectrum-cephalosporin-resistant or carbapenem-resistant (ESC-R/CR) Gram-negative Enterobacteriaceae from three freestanding pediatric centers. Isolates were from urine or other sterile sites of children and adolescents through 21 years of age. Susceptibilities were assayed by microbroth dilution via custom Sensititre plates (Thermo Fisher Scientific). Susceptibility was determined using the Sensititre Vizion® system (Thermo Fisher Scientific). Susceptibility breakpoint criteria were those of the Clinical and Laboratory Standards Institute (CLSI) for 2017, except for colistin (EUCAST 2019). Overall, 87.5% isolates were C/T susceptible (MIC ≤2 µg/ml; MIC50/90, 0.25/4 µg/ml). Susceptibility to C/T was detected more frequently as compared to all other antimicrobials tested except for colistin (95.4%) and meropenem (97.4%). Percent susceptibility to C/T was high for E. coli (91%) and Klebsiella spp. (73.3%). C/T demonstrated good in-vitro activity and high potency against most beta-lactam resistant pediatric Enterobacteriaceae from three geographically diverse U.S. regions.

Emergence of Parechovirus A3 as the Leading Cause of Central Nervous System Infection, Surpassing Any Single Enterovirus Type, in Children in Kansas City, Missouri, USA, from 2007 to 2016.

Picornaviruses, including Enterovirus species A to D (EV) and Parechovirus species A (PeV-A), are the leading reported causes of pediatric central nervous system infections in the United States. We investigated the molecular epidemiology of EV and PeV-A over 10 years in cerebrospinal fluid (CSF) collected from children seen at Children's Mercy-Kansas City (CMKC) from 2007 through 2016. The overall prevalence for EV was 16% (862/5,362) and 7% (271/4,016) for PeV. Among all picornavirus CSF detections, EV was 76%, and PeV-A was 24%. Multiple EV types cocirculated each year, with a total of 31 EV types detected in the 10-year period; the majority belonged to EV-B species (96%). Two PeV-A types were detected; PeV-A3 was the dominant PeV-A type (95%). The top five picornaviruses (PeV-A3, 26%; E30, 11%; E6, 10%; E18, 9%; E9, 7%) in the CSF of infants accounted for two-thirds of all detections, and PeV-A3 was the leading picornavirus detected. Routine testing and reporting of PeV-A in addition to EV, especially in children under 6 months old with acute febrile illnesses, could reduce hospital stays and antibiotic usage.

High-resolution epigenome analysis in nasal samples derived from children with respiratory viral infections reveals striking changes upon SARS-CoV-2 infection.

Background: DNA methylation patterns of the human genome can be modified by environmental stimuli and provide dense information on gene regulatory circuitries. We studied genome-wide DNA methylation in nasal samples from infants (<6 months) applying whole-genome bisulfite sequencing (WGBS) to characterize epigenome response to 10 different respiratory viral infections including SARS-CoV-2. Results: We identified virus-specific differentially methylated regions (vDMR) with human metapneumovirus (hMPV) and SARS-CoV-2 followed by Influenza B (Flu B) causing the weakest vs. strongest epigenome response with 496 vs. 78541 and 14361 vDMR, respectively. We found a strong replication rate of FluB (52%) and SARS-CoV-2 (42%) vDMR in independent samples indicating robust epigenome perturbation upon infection. Among the FluB and SARS-CoV-2 vDMRs, around 70% were hypomethylated and significantly enriched among epithelial cell-specific regulatory elements whereas the hypermethylated vDMRs for these viruses mapped more frequently to immune cell regulatory elements, especially those of the myeloid lineage. The hypermethylated vDMRs were also enriched among genes and genetic loci in monocyte activation pathways and monocyte count. Finally, we perform single-cell RNA-sequencing characterization of nasal mucosa in response to these two viruses to functionally analyze the epigenome perturbations. Which supports the trends we identified in methylation data and highlights and important role for monocytes. Conclusions: All together, we find evidence indicating genetic predisposition to innate immune response upon a respiratory viral infection. Our genome-wide monitoring of infant viral response provides first catalogue of associated host regulatory elements. Assessing epigenetic variation in individual patients may reveal evidence for viral triggers of childhood disease.

Frequency of asymptomatic and symptomatic respiratory virus detection in pediatric hematopoietic cell transplant patients.

Respiratory viral infections are common and can cause significant morbidity and mortality in pediatric patients undergoing hematopoietic cell transplantation (HCT). The prevalence of disease has been primarily identified from retrospective studies using standard-of-care specimens. The incidence of both asymptomatic respiratory viral detection and symptomatic respiratory viral detection in this high-risk population is not well described. We performed longitudinal, active, prospective surveillance in pediatric HCT patients. Subjects underwent weekly midturbinate swabs (MTSs) for the detection of 18 respiratory viruses and subtypes peri-HCT and 100 days post-HCT. Clinical data were obtained from the medical record. From September 2015 to February 2017, 24 children underwent 29 HCT, and 284 MTSs were collected. Forty-two (15%) specimens were virus-positive from 10 (42%) subjects. Specimens from children undergoing allogeneic HCT were more likely to have a virus detected (17% vs 8%, P = .04) compared with specimens from children undergoing autologous HCT. Sixteen (38%) detections were not associated with symptoms. Almost half (8/17) of the unique viral infections occurred during the HCT hospitalization after a negative specimen, suggesting nosocomial acquisition, and preceded detection from a clinical specimen. Rhinovirus, the most common virus detected, was the only virus detected in 33 (81%) virus-positive specimens; only 11 (33%) rhinovirus detections were asymptomatic. Asymptomatic detection of coronavirus and bocavirus occurred. Asymptomatic respiratory virus detection occurred in more than one-third of the children undergoing HCT. The acquisition of respiratory viruses during HCT hospitalization suggests nosocomial acquisition. Early detection of respiratory viruses during asymptomatic periods could have infection prevention and treatment implications.

Comparative analysis of Four sample-to-answer influenza A/B and RSV nucleic acid amplification assays using adult respiratory specimens.

BACKGROUND: The use of Sample-to-answer (STA) platforms for the detection of influenza A/B and respiratory syncytial virus (RSV) have greatly improved patient care. These diagnostic assays based on nucleic acid amplification are rapid, accurate and relatively easy to perform. OBJECTIVES: We compared four such platforms for detecting FluA, FluB, and RSV from adult respiratory specimens: Hologic Panther Fusion® Flu A/B/RSV (Fusion), Cobas® Influenza A/B & RSV (Liat), Luminex Aries® Flu A/B & RSV (Aries), and Diasorin SimplexaTM Flu A/B & RSV (Simplexa). STUDY DESIGN: Nasopharyngeal (NP) swabs (n = 224) from adults were tested on these platforms and results were compared to Center for Disease Control and Prevention recommended real-time RT-PCR assay for influenza A/B and RSV. Subtyping for FluA and FluB was performed for discrepant analysis where applicable. RESULTS: Of the 82 FluA, 26 FluB, 15 RSV-positive specimens tested, the positive and negative percentage agreements (PPA and NPA respectively) for FluA detection were 100/100 (Fusion), 95.1/100 (Liat), 92.5/100 (Aries), and 84.1/99.3 (Simplexa); PPA and NPA for FluB detection were 92.3/99.5 (Fusion), 96/99.5 (Liat), 100/99.5 (Aries), and 80.8/100 (Simplexa); and for RSV detection were 100/100 (Fusion), 100/100 (Liat), 88.6/99.5 (Aries), and 73.3/100 (Simplexa). 82 confirmed FluA included 23 pH1N1 and 57 H3N2 strains with 2 strains remaining untyped. Of the 26 confirmed FluB, 25 were of the Yamagata lineage and 1 of unknown lineage. CONCLUSION: Only 2 STA platforms demonstrated >95% PPA for the detection of all three targets while all the 4 platforms demonstrated >95% NPA for FluA, FluB and RSV.

Comparison of Six Sample-to-Answer Influenza A/B and Respiratory Syncytial Virus Nucleic Acid Amplification Assays Using Respiratory Specimens from Children.

The rapid and accurate detection of influenza A virus (FluA), influenza B virus (FluB), and respiratory syncytial virus (RSV) improves patient care. Sample-to-answer (STA) platforms based on nucleic acid amplification and detection of these viruses are simple, automated, and accurate. We compared six such platforms for the detection of FluA, FluB, and RSV: Cepheid GeneXpert Xpress Flu/RSV (Xpert), Hologic Panther Fusion Flu A/B/RSV (Fusion), Cobas influenza A/B & RSV (Liat), Luminex Aries Flu A/B & RSV (Aries), BioFire FilmArray respiratory panel (RP), and Diasorin Simplexa Flu A/B & RSV (Simplexa). Nasopharyngeal (NP) swab specimens (n = 225) from children previously tested by RP were assessed on these platforms. The results were compared to those of the Centers for Disease Control and Prevention (CDC)-developed real-time reverse transcription-PCR (rRT-PCR) assay for influenza A/B viruses and RSV. Subtyping for FluA and FluB was performed for discrepant analysis where applicable. The percent sensitivities/specificities for FluA detection were 100/100 (Fusion), 98.6/99.3 (Xpert), 100/100 (Liat), 98.6/100 (Aries), 98.6/100 (Simplexa), and 100/100 (RP). The percent sensitivities/specificities for FluB detection were 100/100 (Fusion), 97.9/99.4 (Xpert), 97.9/98.3 (Liat), 93.7/99.4 (Aries), 85.4/99.4 (Simplexa), and 95.8/97.7 (RP); and those for RSV detection were 98.1/99.4 (Xpert), 98.1/99.4 (Liat), 96.3/100 (Fusion), 94.4/100 (Aries), 87/94.4 (Simplexa), and 94.4/100 (RP). The 75 strains confirmed to be FluA included 29 pH1N1, 39 H3N2, 4 sH1N1, and 3 untyped strains. The 48 strains confirmed to be FluB included 33 strains of the Yamagata lineage, 13 of the Victoria lineage, 1 of both the Yamagata and Victoria lineages, and 1 of an unknown lineage. All six STA platforms demonstrated >95% sensitivity for FluA detection, while three platforms (Fusion, Xpert, and Liat) demonstrated >95% sensitivity for FluB and RSV detection.

De Novo Pyrimidine Biosynthesis Connects Cell Integrity to Amphotericin B Susceptibility in Cryptococcus neoformans.

The use of amphotericin B (AmB) in conjunction with 5-fluorocytosine (5-FC) is known to be the optimal therapy for treating cryptococcosis, but the mechanism by which 5-FC synergizes with AmB is unknown. In this study, we generated a Cryptococcus neoformans ura1Δ mutant lacking dihydroorotate dehydrogenase (DHODH), which demonstrated temperature-sensitive growth due to a defect in cell integrity and sensitivity to cell wall-damaging agents. In addition, sensitivity to AmB was greatly increased. Inclusion of uracil or uridine in the medium did not suppress the cell wall or AmB phenotype, whereas complementation with the wild-type URA1 gene complemented the mutant phenotype. As a measure of membrane accessibility, we assayed the rate of association of the lipid-binding dye 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and saw more rapid association in the ura1Δ mutant. We likewise saw an increased rate of DiOC6 association in other AmB-sensitive mutants, including a ura- spontaneous URA5 mutant made by 5-fluoroorotic acid (5-FOA) selection and a bck1Δ mutant defective in cell integrity signaling. Similar results were also obtained by using a specific plasma membrane-binding CellMask live stain, with cell integrity mutants that exhibited increased and faster association of the dye with the membrane. Chitin synthase mutants (chs5Δ and chs6Δ) that lack any reported cell wall defects, in turn, demonstrate neither any increased susceptibility to AmB nor a greater accessibility to either of the dyes. Finally, perturbation of the cell wall of the wild type by treatment with the ß-1,6-glucan synthase inhibitor caspofungin was synergistic with AmB in vitro. IMPORTANCE Synergy between AmB and nucleotide biosynthetic pathways has been documented, but the mechanism of this interaction has not been delineated. Results from this study suggest a correlation between uridine nucleotide biosynthesis and cell integrity likely mediated through the pool of nucleotide-sugar conjugates, which are precursor molecules for both capsule and cell wall of C. neoformans. Thus, we propose a mechanism by which structural defects in the cell wall resulting from perturbation of pyrimidine biosynthesis allow faster and increased penetration of AmB molecules into the cell membrane. Overall, our work demonstrates that impairment of pyrimidine biosynthesis in C. neoformans could be a potential target for antifungal therapy, either alone or in combination with AmB.