Children and youth infected with SARS-CoV-2 have milder disease than do adults and, even among those with the recently described multi-system inflammatory syndrome (MIS-C), mortality is rare. The reasons for the differences in clinical manifestations are unknown, but suggest that age-dependent factors may modulate the anti-viral immune response. We compared cytokine, humoral, and cellular immune responses in pediatric (children and youth, age < 24 years) (n=65) and adult (n=60) patients with COVID-19 at a metropolitan hospital system in New York City. The pediatric patients had a shorter length of stay, decreased requirement for mechanical ventilation and lower mortality compared to adults. The serum concentrations of IL-17A and IFN-γ, but not TNF-α or IL-6, were inversely related to age. Adults mounted a more robust T cell response to the viral spike protein compared to pediatric patients as evidenced by increased expression of CD25+ on CD4+ T cells and the frequency of IFN-γ+CD4+ T cells. Moreover, serum neutralizing antibody titers and antibody-dependent cellular phagocytosis were higher in adults compared to pediatric COVID-19 patients. The neutralizing antibody titer correlated positively with age and negatively with IL-17A and IFN-γ serum concentrations. There were no differences in anti-spike protein antibody titers to other human coronaviruses. Together these findings demonstrate that the poor outcome in hospitalized adults with COVID-19 compared to children may not be attributable to a failure to generate adaptive immune responses.

Background: COVID-19 is more benign in children compared to adults for unknown reasons. This contrasts with other respiratory viruses where disease manifestations are often more severe in children. We hypothesize that a more robust early innate immune response to SARS-CoV-2 protects against severe disease. Methods: Clinical outcomes, SARS-CoV-2 viral copies and cellular gene expression were compared in nasopharyngeal swabs obtained at the time of presentation to the Emergency Department from 12 children and 27 adults using bulk RNA sequencing and quantitative reverse transcription PCR. Total protein, cytokines and anti-SARS-CoV-2 IgG and IgA were quantified in nasal fluid.Results: SARS-CoV-2 copies, ACE2 and TMPRSS2 gene expression were similar in children and adults, but children displayed higher expression of genes associated with interferon signaling, NLRP3 inflammasome, and other innate pathways. Higher levels of IFN-2, IFN-, IP-10, IL-8, and IL-1 protein were detected in nasal fluid in children versus adults. Children also expressed higher levels of genes associated with immune cells whereas expression of those associated with epithelial cells did not differ in children versus adults. Anti-SARS-CoV-2 IgA and IgG were detected at similar levels in nasal fluid from both groups. None of the children required supplemental oxygen whereas 7 adults did (p=0.03); four adults died.Conclusions: These findings provide direct evidence of a more vigorous early mucosal immune response in children compared to adults and suggest that this contributes to favorable clinical outcomes.

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Candida auris is an emerging multi-drug resistant yeast that causes systemic infections. Here we show that C. auris undergoes replicative aging (RA) that results from asymmetric cell division and causes phenotypic differences between mother and daughter cells similar to other pathogenic yeasts. Importantly, older C. auris cells (10 generations) exhibited higher tolerance to fluconazole (FLC), micafungin, 5- flucytosine and amphotericin B compared to younger (0–3 generation) cells. Increased FLC tolerance was associated with increased Rhodamine 6G (R6G) efflux and therapeutic failure of FLC in a Galleria infection model. The higher efflux in the older cells correlated with overexpression of the efflux pump encoding gene CDR1 (4-fold). In addition, 8-fold upregulation of the azole target encoding gene ERG11 was noted in the older cells. Analysis of genomic DNA from older cells by qPCR indicates that transient gene duplication of CDR1 and ERG11 causes the observed age-dependent enhanced FLC tolerance in C. auris strains. Furthermore, older cells exhibited a thickened cell wall, decreased neutrophil killing (24% vs 50%), increased epithelial cell adhesion (31.6% vs 17.8%) and upregulation of adhesin protein Als5p. Thus, this study demonstrates that transient gene duplication can occur during RA, causing increased FLC tolerance in old C. auris cells.

Cryptococcus neoformans is an encapsulated fungal pathogen that causes meningoencephalitis. There are no prophylactic tools for cryptococcosis. Previously, our group showed that a C. neoformans mutant lacking the gene encoding sterylglucosidase (Δsgl1) induced protection in both immunocompetent and immunocompromised murine models of cryptococcosis. Since sterylglucosidase catalyzes degradation of sterylglucosides (SGs), accumulation of this glycolipid could be responsible for protective immunity. In this study, we analyzed whether the activity of SGs is sufficient for the protective effect induced by the Δsgl1 strain. We observed that the accumulation of SGs impacted several properties of the main polysaccharide that composes the fungal capsule, glucuronoxylomannan (GXM). We therefore used genetic manipulation to delete the SGL1 gene in the acapsular mutant Δcap59 to generate a double mutant (strain Δcap59/Δsgl1) that was shown to be nonpathogenic and cleared from the lung of mice within 7 days post-intranasal infection. The inflammatory immune response triggered by the Δcap59/Δsgl1 mutant in the lung differed from the response seen with the other strains. The double mutant did not induce protection in a vaccination model, suggesting that SG-related protection requires the main capsular polysaccharide. Finally, GXM-containing extracellular vesicles (EVs) enriched in SGs delayed the acute lethality of Galleria mellonella against C. neoformans infection. These studies highlighted a key role for GXM and SGs in inducing protection against a secondary cryptococcal infection, and, since EVs notoriously contain GXM, these results suggest the potential use of Δsgl1 EVs as a vaccination strategy for cryptococcosis. IMPORTANCE The number of deaths from cryptococcal meningitis is around 180,000 per year. The disease is the second leading cause of mortality among individuals with AIDS. Antifungal treatment is costly and associated with adverse effects and resistance, evidencing the urgency of development of both therapeutic and prophylactic tools. Here we demonstrate the key roles of polysaccharide- and glycolipid-containing structures in a vaccination model to prevent cryptococcosis.

Similar to other yeasts, the human pathogen Candida glabrata ages when it undergoes asymmetric, finite cell divisions, which determines its replicative lifespan. We sought to investigate if and how aging changes resilience of C. glabrata populations in the host environment. Our data demonstrate that old C. glabrata are more resistant to hydrogen peroxide and neutrophil killing, whereas young cells adhere better to epithelial cell layers. Consequently, virulence of old compared to younger C. glabrata cells is enhanced in the Galleria mellonella infection model. Electron microscopy images of old C. glabrata cells indicate a marked increase in cell wall thickness. Comparison of transcriptomes of old and young C. glabrata cells reveals differential regulation of ergosterol and Hog pathway associated genes as well as adhesion proteins, and suggests that aging is accompanied by remodeling of the fungal cell wall. Biochemical analysis supports this conclusion as older cells exhibit a qualitatively different lipid composition, leading to the observed increased emergence of fluconazole resistance when grown in the presence of fluconazole selection pressure. Older C. glabrata cells accumulate during murine and human infection, which is statistically unlikely without very strong selection. Therefore, we tested the hypothesis that neutrophils constitute the predominant selection pressure in vivo. When we altered experimentally the selection pressure by antibody-mediated removal of neutrophils, we observed a significantly younger pathogen population in mice. Mathematical modeling confirmed that differential selection of older cells is sufficient to cause the observed demographic shift in the fungal population. Hence our data support the concept that pathogenesis is affected by the generational age distribution of the infecting C. glabrata population in a host. We conclude that replicative aging constitutes an emerging trait, which is selected by the host and may even play an unanticipated role in the transition from a commensal to a pathogen state.

Background The clinical and public health utility of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serologic testing requires a better understanding of the dynamics of the humoral response to infection. Objective To track seroconversion of IgG and IgM antibodies in patients with SARS-CoV-2 infection and its association with patient and clinical factors and outcomes. Methods Residual patient specimens were analyzed on the Abbott ARCHITECT i2000 instrument using the Abbott SARS-CoV-2 IgG assay and prototype SARS-CoV-2 IgM assay. Age, sex, comorbidities, symptom onset date, mortality, and specimen collection date were obtained from electronic medical records. Results 359 longitudinal samples were collected from 89 hospitalized patients 0-82 days post-symptom onset. 51.7% of patients developed IgG and IgM antibodies simultaneously; 32.8% seroconverted for IgM before IgG. On average, patients seroconverted for IgG by 8 days and for IgM by 7 days post-symptom onset. All patients achieved IgG seropositivity by 19 days and IgM seropositivity by 17 days. Median time to IgG and IgM seroconversion was prolonged and initial levels of IgG were lower in immunocompromised patients and patients <65 years of age compared to immune competent patients and those ≥65 years of age. Immunocompromised patients also had persistently lower levels of IgM that peaked on day 17.6 and decreased thereafter compared to immune competent patients. IgM seroconversion in patients who died reached significantly higher levels later after symptom onset than in those who recovered. Conclusions SARS-CoV-2 infected patients have similar time to seroconversion for IgG and IgM. However, differences in immune status and age alter time to seroconversion. These results may help guide serologic testing application in COVID-19 management.

The ability to detect SARS-CoV-2 in the upper respiratory tract ceases after two to three weeks post symptom onset in most patients. In contrast, SARS-CoV-2 can be detected in the stool of some patients for greater than four weeks suggesting that stool may hold utility as an additional source for diagnosis. We validated the Cepheid Xpert Xpress SARS-CoV-2 and Hologic Panther Fusion real-time RT-PCR assays for detection of viral RNA in stool specimens and compared performance. We utilized remnant stool specimens (n=79) from 77 patients with gastrointestinal symptoms. Forty-eight patients had PCR-confirmed COVID-19 and 29 were either nasopharyngeal/oropharyngeal PCR negative or presented for reasons unrelated to COVID-19 and were not tested. Positive percent agreement between the Cepheid and Hologic assays was 93% (95% CI: 81.1%-98.2%) and negative percent agreement was 96% (95% CI: 89%-0.99%). Four discrepant specimens (Cepheid positive only, n=2; Hologic positive only, n=2) exhibited average Ct values of >37 for the targets detected. Of the 48 patients with PCR-confirmed COVID-19, 23 were positive by both assays (47.9%). For the negative patient group, 2/29 were positive by both assays (6.9%). The two stool PCR positive, nasopharyngeal/oropharyngeal PCR negative patients were SARS-CoV-2 IgG positive. Our results demonstrate acceptable agreement between two commercially available molecular assays and support the use of stool PCR to confirm diagnosis when SARS-CoV-2 is undetectable in the upper respiratory tract.