Disseminated Coccidioidomycosis Treated with Interferon-γ and Dupilumab.

We describe a case of life-threatening disseminated coccidioidomycosis in a previously healthy child. Like most patients with disseminated coccidioidomycosis, this child had no genomic evidence of any known, rare immune disease. However, comprehensive immunologic testing showed exaggerated production of interleukin-4 and reduced production of interferon-γ. Supplementation of antifungal agents with interferon-γ treatment slowed disease progression, and the addition of interleukin-4 and interleukin-13 blockade with dupilumab resulted in rapid resolution of the patient's clinical symptoms. This report shows that blocking of type 2 immune responses can treat infection. This immunomodulatory approach could be used to enhance immune clearance of refractory fungal, mycobacterial, and viral infections. (Supported by the Jeffrey Modell Foundation and the National Institutes of Health.).

No infectious SARS-CoV-2 in breast milk from a cohort of 110 lactating women.

BACKGROUND: Genomic RNA of severe acute respiratory syndrome-associated coronavirus type 2 (SARS-CoV-2) has been detected in the breast milk of lactating women, but its pathological significance has remained uncertain due to the small size of prior studies. METHODS: Breast milk from 110 lactating women was analyzed by reverse transcription-polymerase chain reaction (285 samples) and viral culture (160 samples). Those containing SARS-CoV-2 viral RNA (vRNA) were examined for the presence of subgenomic RNA (sgRNA), a putative marker of infectivity. RESULTS: Sixty-five women had a positive SARS-CoV-2 diagnostic test, 9 had symptoms but negative diagnostic tests, and 36 symptomatic women were not tested. SARS-CoV-2 vRNA was detected in the milk of 7 (6%) women with either a confirmed infection or symptomatic illness, including 6 of 65 (9%) women with a positive SARS-CoV-2 diagnostic test. Infectious virus was not detected in any culture and none had detectable sgRNA. In control experiments, infectious SARS-CoV-2 could be cultured after addition to breastmilk despite several freeze-thaw cycles, as it occurs in the storage and usage of human milk. CONCLUSIONS: SARS-CoV-2 RNA can be found infrequently in the breastmilk after recent infection, but we found no evidence that breastmilk contains an infectious virus or that breastfeeding represents a risk factor for transmission of infection to infants. IMPACT: This article goes beyond prior small studies to provide evidence that infectious SARS-CoV-2 is not present in the milk of lactating women with recent infection, even when SARS-CoV-2 RNA is detected. Recent SARS-CoV-2 infection or detection of its RNA in human milk is not a contraindication to breastfeeding.

Enteroviruses in the early 21st century: new manifestations and challenges.

PURPOSE OF REVIEW: Enteroviruses cause a wide variety of diseases with neurologic, respiratory, skin, and gastrointestinal findings. The purpose of this review is to clarify changes in the classification of enteroviruses, provide information about recent disease outbreaks, and to summarize progress toward the treatment and prevention of these infections. RECENT FINDINGS: Enteroviruses are now classified into four distinct species. New variants of coxsackievirus B1, enterovirus-A71, and enterovirus-D68 (EV-D68) have emerged as causes of recent outbreaks in the United States and other countries, including more severe disease manifestations than previously described. EV-D68 now commonly circulates in the United States, and has been linked to severe respiratory disease and associated with acute flaccid myelitis (AFM). Overcoming enormous political and logistical challenges, fewer than 100 cases of polio have been reported in 2015, and the initiation of 'endgame' strategies appears imminent. Unfortunately, treatment for enterovirus infections remains supportive, although the recently completed pleconaril trial in newborns suggests that antiviral therapy may reduce mortality in neonatal disease. SUMMARY: Clinicians should be aware of the respiratory and neurological manifestations associated with EV-D68 and the potential for severe disease seen with other recently described enterovirus variants. Healthcare professionals should recognize the utility of rapid diagnostic methods and progress toward prevention and treatment of enterovirus infections.

Leishmaniasis Gone Viral: Social Media and an Outbreak of Cutaneous Leishmaniasis.

This report details how social media communication was used in a group of teens to diagnose cutaneous leishmaniasis that they acquired during a trip to Israel. Their posts quickly brought the cluster to the attention of the teens and their parents, leading to prompt recognition of the true etiology of their lesions and appropriate treatment.

Discovery of Structurally Diverse Small-Molecule Compounds with Broad Antiviral Activity against Enteroviruses.

Antiviral drugs do not currently exist for the treatment of enterovirus infections, which are often severe and potentially life-threatening. We conducted high-throughput molecular screening and identified a structurally diverse set of compounds that inhibit the replication of coxsackievirus B3, a commonly encountered enterovirus. These compounds did not interfere with the function of the viral internal ribosome entry site or with the activity of the viral proteases, but they did drastically reduce the synthesis of viral RNA and viral proteins in infected cells. Sequence analysis of compound-resistant mutants suggests that the viral 2C protein is targeted by most of these compounds. These compounds demonstrated antiviral activity against a panel of the most commonly encountered enteroviruses and thus represent potential leads for the development of broad-spectrum anti-enteroviral drugs.

Serological misdiagnosis of acute liver failure associated with echovirus 25 due to immunological similarities to hepatitis A virus and prozone effect.

We describe a case of acute liver failure caused by echovirus 25 (E25) in a previously healthy 2-year-old boy. Initial serological studies were consistent with hepatitis A virus (HAV), with prozone phenomenon. The similarity of E25 to HAV may obscure accurate diagnosis in some cases of hepatitis.

A broad-spectrum antiviral targeting entry of enveloped viruses.

We describe an antiviral small molecule, LJ001, effective against numerous enveloped viruses including Influenza A, filoviruses, poxviruses, arenaviruses, bunyaviruses, paramyxoviruses, flaviviruses, and HIV-1. In sharp contrast, the compound had no effect on the infection of nonenveloped viruses. In vitro and in vivo assays showed no overt toxicity. LJ001 specifically intercalated into viral membranes, irreversibly inactivated virions while leaving functionally intact envelope proteins, and inhibited viral entry at a step after virus binding but before virus-cell fusion. LJ001 pretreatment also prevented virus-induced mortality from Ebola and Rift Valley fever viruses. Structure-activity relationship analyses of LJ001, a rhodanine derivative, implicated both the polar and nonpolar ends of LJ001 in its antiviral activity. LJ001 specifically inhibited virus-cell but not cell-cell fusion, and further studies with lipid biosynthesis inhibitors indicated that LJ001 exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes with reparative capacity. In sum, our data reveal a class of broad-spectrum antivirals effective against enveloped viruses that target the viral lipid membrane and compromises its ability to mediate virus-cell fusion.

A Clinicopathological Categorization System for Clinical Research in Coccidioidomycosis.

A wide array of clinical manifestations follow infection with Coccidioides immitis or Coccidioides posadasii, ranging from asymptomatic infection to life-threatening pulmonary disease or extrapulmonary dissemination and meningitis. Epidemiological studies require consistent definitions of cases and their comparative clinical features. Understanding host and pathogen determinants of the severity of coccidioidomycosis also requires that specific clinical features (such as coccidioidal meningitis) and their overlap be precisely defined and quantified. Here we propose a system for categorization of outcomes of coccidioidomycosis in individuals who are not overtly immunocompromised that harmonizes clinical assessments during translational research of this increasingly common disease.

Persistent memory despite rapid contraction of circulating T Cell responses to SARS-CoV-2 mRNA vaccination.

Introduction: While antibodies raised by SARS-CoV-2 mRNA vaccines have had compromised efficacy to prevent breakthrough infections due to both limited durability and spike sequence variation, the vaccines have remained highly protective against severe illness. This protection is mediated through cellular immunity, particularly CD8+ T cells, and lasts at least a few months. Although several studies have documented rapidly waning levels of vaccine-elicited antibodies, the kinetics of T cell responses have not been well defined. Methods: Interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot) assay and intracellular cytokine staining (ICS) were utilized to assess cellular immune responses (in isolated CD8+ T cells or whole peripheral blood mononuclear cells, PBMCs) to pooled peptides spanning spike. ELISA was performed to quantitate serum antibodies against the spike receptor binding domain (RBD). Results: In two persons receiving primary vaccination, tightly serially evaluated frequencies of anti-spike CD8+ T cells using ELISpot assays revealed strikingly short-lived responses, peaking after about 10 days and becoming undetectable by about 20 days after each dose. This pattern was also observed in cross-sectional analyses of persons after the first and second doses during primary vaccination with mRNA vaccines. In contrast, cross-sectional analysis of COVID-19-recovered persons using the same assay showed persisting responses in most persons through 45 days after symptom onset. Cross-sectional analysis using IFN-γ ICS of PBMCs from persons 13 to 235 days after mRNA vaccination also demonstrated undetectable CD8+ T cells against spike soon after vaccination, and extended the observation to include CD4+ T cells. However, ICS analyses of the same PBMCs after culturing with the mRNA-1273 vaccine in vitro showed CD4+ and CD8+ T cell responses that were readily detectable in most persons out to 235 days after vaccination. Discussion: Overall, we find that detection of spike-targeted responses from mRNA vaccines using typical IFN-γ assays is remarkably transient, which may be a function of the mRNA vaccine platform and an intrinsic property of the spike protein as an immune target. However, robust memory, as demonstrated by capacity for rapid expansion of T cells responding to spike, is maintained at least several months after vaccination. This is consistent with the clinical observation of vaccine protection from severe illness lasting months. The level of such memory responsiveness required for clinical protection remains to be defined.

IFN-γ-mediated control of SARS-CoV-2 infection through nitric oxide.

Introduction: The COVID-19 pandemic has highlighted the need to identify mechanisms of antiviral host defense against SARS-CoV-2. One such mediator is interferon-g (IFN-γ), which, when administered to infected patients, is reported to result in viral clearance and resolution of pulmonary symptoms. IFN-γ treatment of a human lung epithelial cell line triggered an antiviral activity against SARS-CoV-2, yet the mechanism for this antiviral response was not identified. Methods: Given that IFN-γ has been shown to trigger antiviral activity via the generation of nitric oxide (NO), we investigated whether IFN-γ induction of antiviral activity against SARS-CoV-2 infection is dependent upon the generation of NO in human pulmonary epithelial cells. We treated the simian epithelial cell line Vero E6 and human pulmonary epithelial cell lines, including A549-ACE2, and Calu-3, with IFN-γ and observed the resulting induction of NO and its effects on SARS-CoV-2 replication. Pharmacological inhibition of inducible nitric oxide synthase (iNOS) was employed to assess the dependency on NO production. Additionally, the study examined the effect of interleukin-1b (IL-1ß) on the IFN-g-induced NO production and its antiviral efficacy. Results: Treatment of Vero E6 cells with IFN-γ resulted in a dose-responsive induction of NO and an inhibitory effect on SARS-CoV-2 replication. This antiviral activity was blocked by pharmacologic inhibition of iNOS. IFN-γ also triggered a NO-mediated antiviral activity in SARS-CoV-2 infected human lung epithelial cell lines A549-ACE2 and Calu-3. IL-1ß enhanced IFN-γ induction of NO, but it had little effect on antiviral activity. Discussion: Given that IFN-g has been shown to be produced by CD8+ T cells in the early response to SARS-CoV-2, our findings in human lung epithelial cell lines, of an IFN-γ-triggered, NO-dependent, links the adaptive immune response to an innate antiviral pathway in host defense against SARS-CoV-2. These results underscore the importance of IFN-γ and NO in the antiviral response and provide insights into potential therapeutic strategies for COVID-19.

Fluoxetine is a potent inhibitor of coxsackievirus replication.

No antiviral drugs currently exist for the treatment of enterovirus infections, which are often severe and potentially life threatening. Molecular screening of small molecule libraries identified fluoxetine, a selective serotonin reuptake inhibitor, as a potent inhibitor of coxsackievirus replication. Fluoxetine did not interfere with either viral entry or translation of the viral genome. Instead, fluoxetine and its metabolite norfluoxetine markedly reduced the synthesis of viral RNA and protein. In view of its favorable pharmacokinetics and safety profile, fluoxetine warrants additional study as a potential antiviral agent for enterovirus infections.

Dominant CD8+ T Cell Nucleocapsid Targeting in SARS-CoV-2 Infection and Broad Spike Targeting From Vaccination.

CD8+ T cells have key protective roles in many viral infections. While an overall Th1-biased cellular immune response against SARS-CoV-2 has been demonstrated, most reports of anti-SARS-CoV-2 cellular immunity have evaluated bulk T cells using pools of predicted epitopes, without clear delineation of the CD8+ subset and its magnitude and targeting. In recently infected persons (mean 29.8 days after COVID-19 symptom onset), we confirm a Th1 bias (and a novel IL-4-producing population of unclear significance) by flow cytometry, which does not correlate to antibody responses against the receptor binding domain. Evaluating isolated CD8+ T cells in more detail by IFN-γ ELISpot assays, responses against spike, nucleocapsid, matrix, and envelope proteins average 396, 901, 296, and 0 spot-forming cells (SFC) per million, targeting 1.4, 1.5, 0.59, and 0.0 epitope regions respectively. Nucleocapsid targeting is dominant in terms of magnitude, breadth, and density of targeting. The magnitude of responses drops rapidly post-infection; nucleocapsid targeting is most sustained, and vaccination selectively boosts spike targeting. In SARS-CoV-2-naïve persons, evaluation of the anti-spike CD8+ T cell response soon after vaccination (mean 11.3 days) yields anti-spike CD8+ T cell responses averaging 2,463 SFC/million against 4.2 epitope regions, and targeting mirrors that seen in infected persons. These findings provide greater clarity on CD8+ T cell anti-SARS-CoV-2 targeting, breadth, and persistence, suggesting that nucleocapsid inclusion in vaccines could broaden coverage and durability.

New Delhi metallo-ß-lactamase (NDM-1)-producing Klebsiella pneumoniae: case report and laboratory detection strategies.

The spread of antimicrobial resistance among Enterobacteriaceae is a significant clinical threat. We report the first case of an Enterobacteriaceae strain harboring the NDM-1 metallo-ß-lactamase in a pediatric patient in the United States. We describe strategies for the detection of this novel resistance mechanism encountered in an isolate of Klebsiella pneumoniae.

No Evidence of Infectious SARS-CoV-2 in Human Milk: Analysis of a Cohort of 110 Lactating Women.

BACKGROUND: SARS-CoV-2 infections of infants and toddlers are usually mild but can result in life-threatening disease. SARS-CoV-2 RNA been detected in the breast milk of lactating women, but the potential role of breastfeeding in transmission to infants has remained uncertain. METHODS: Breast milk specimens were examined for the presence of the virus by RT-PCR and/or culture. Specimens that contained viral RNA (vRNA) were examined for the presence of subgenomic coronavirus RNA (sgRNA), a putative marker of infectivity. Culture methods were used to determine the thermal stability of SARS-CoV-2 in human milk. RESULTS: Breast milk samples from 110 women (65 confirmed with a SARS-CoV-2 diagnostic test, 36 with symptoms but without tests, and 9 with symptoms but a negative SARS-CoV-2 diagnostic test) were tested by RT-PCR (285 samples) and/or viral culture (160 samples). Although vRNA of SARS-CoV-2 was detected in the milk of 7 of 110 (6%) women with either a confirmed infection or symptomatic illness, and in 6 of 65 (9%) of women with a positive SARS-CoV-2 diagnostic test, virus was not detected in any culture. None of the 7 milk specimens with detectable vRNA contained sgRNA. Notably, when artificially added to human milk in control experiments, infectious SARS-CoV-2 could be cultured despite several freeze-thaw cycles, as occurs in the storage and usage of human milk. CONCLUSIONS: SARS-CoV-2 RNA can be found infrequently in the breastmilk of women with recent infection, but we found no evidence that breastmilk contains infectious virus or that breastfeeding represents a risk factor for transmission of infection to infants. KEY POINTS: Question: SARS-CoV-2 RNA has been detected in a small number of human milk samples collected from recently infected women. The role of breastfeeding in transmission of the virus to infants has remained uncertain due to the small number of specimens analyzed in any study published thus far.Findings: In a total study group of 110 women, SARS-CoV-2 RNA was detected in milk from 6 of 65 women (9.2%) with recent confirmed infection. Neither infectious virus nor subgenomic RNA (a marker of virus infectivity) were detected in any of the samples.Meaning: We found no evidence that infectious SARS-CoV-2 is present milk from recently infected women, even if SARS-CoV-2 PCR tests are positive, providing reassurance of the safety of breastfeeding.

The utility of echocardiography in paediatric patients with musculoskeletal infections and bacteremia.

PURPOSE: The clinical utility of echocardiography in the setting of a positive blood culture in paediatric patients presenting with osteomyelitis (OM) and/or septic arthritis (SA). METHODS: Retrospective review between 2013 and 2019: Patients < 18 years with OM, SA or combined infection (OM+SA) were included. Patients were excluded for immunodeficiency, loss of follow-up or penetrating infection. Charts with positive blood cultures were reviewed for echocardiography on that admission. Demographic variables were compared utilizing the Student's t-test and Fisher's exact test. A multivariable linear regression model was constructed to examine the association between echocardiography and length of stay, controlling for age, sex, fever, white blood cell (WBC) on admission, antibiotic administration and surgery performed. RESULTS: Of 157 patients with OM, SA or combined infection, 44 had a positive blood culture. In all, 26 had an echocardiogram, and none showed endocarditis. Echocardiography was independently associated with a 6.2-day length of stay increase. WBC count and surgical intervention demonstrated a trend toward significance in length of stay, with each WBC unit increase associated with a 0.53-day increase. Surgical intervention was associated with an average 6.3-day length of stay decrease. CONCLUSION: No patient had a positive echocardiogram, and no changes in management were initiated. However, an echocardiogram increased stay by 6.2 days. In addition to costs associated with increased stay, patients were billed between $1460 and $1700 per echocardiogram. The utility of echocardiograms in the setting of bacteremia associated with musculoskeletal infections in the paediatric population should be re-examined, and guidelines should be updated to reflect the cost-benefit analysis. LEVEL OF EVIDENCE: III.

Pharmacokinetics and Safety of a Raltegravir-Containing Regimen in Children Aged 4 Weeks to 2 Years Living With Human Immunodeficiency Virus and Receiving Rifampin for Tuberculosis.

Pharmacological interactions limit treatment options for children living with human immunodeficiency virus (HIV) and tuberculosis (TB). We found that 12 mg/kg twice daily raltegravir chewable tablets (administered after crushing) safely achieved pharmacokinetic targets in children living with HIV aged 4 weeks to <2 years receiving concurrent rifampin to treat TB. CLINICAL TRIALS REGISTRATION: NCT01751568.

Primary, Recall, and Decay Kinetics of SARS-CoV-2 Vaccine Antibody Responses.

Studies of two SARS-CoV-2 mRNA vaccines suggested that they yield ∼95% protection from symptomatic infection at least short-term, but important clinical questions remain. It is unclear how vaccine-induced antibody levels quantitatively compare to the wide spectrum induced by natural SARS-CoV-2 infection. Vaccine response kinetics and magnitudes in persons with prior COVID-19 compared to virus-naïve persons are not well-defined. The relative stability of vaccine-induced versus infection-induced antibody levels is unclear. We addressed these issues with longitudinal assessments of vaccinees with and without prior SARS-CoV-2 infection using quantitative enzyme-linked immunosorbent assay (ELISA) of anti-RBD antibodies. SARS-CoV-2-naïve individuals achieved levels similar to mild natural infection after the first vaccination; a second dose generated levels approaching severe natural infection. In persons with prior COVID-19, one dose boosted levels to the high end of severe natural infection even in those who never had robust responses from infection, increasing no further after the second dose. Antiviral neutralizing assessments using a spike-pseudovirus assay revealed that virus-naïve vaccinees did not develop physiologic neutralizing potency until the second dose, while previously infected persons exhibited maximal neutralization after one dose. Finally, antibodies from vaccination waned similarly to natural infection, resulting in an average of ∼90% loss within 90 days. In summary, our findings suggest that two doses are important for quantity and quality of humoral immunity in SARS-CoV-2-naïve persons, while a single dose has maximal effects in those with past infection. Antibodies from vaccination wane with kinetics very similar to that seen after mild natural infection; booster vaccinations will likely be required.

Evaluation of SARS-CoV-2 in Breastmilk from 18 Infected Women.

To The Editor, Currently, the U.S. Centers for Disease Control and Prevention, American Academy of Pediatrics and the World Health Organization advise that women who are infected with SARS-CoV-2 may choose to breastfeed with appropriate protections to prevent transmission of the virus through respiratory droplets. However, the potential for exposure to SARS-CoV-2 through breastfeeding is currently unknown. To date, case reports on breastmilk samples from a total of 24 SARS-CoV-2-infected women have been published. Of those, viral RNA was detected in ten breastmilk samples from four women. In some but not all cases, environmental contamination as the source of the virus or retrograde flow from an infected infant could not be ruled out. We established a quantitative RT-PCR assay for SARS-CoV-2 in breastmilk with a limit of detection of 250 copies per mL and validated it by spiking breastmilk from uninfected women with known amounts of viral RNA. In addition, we established tissue culture methods to detect replication-competent SARS-CoV-2 in breastmilk. No viral RNA nor culturable virus was detected after Holder pasteurization of breastmilk samples that had been spiked with replication-competent SARS-CoV-2 (see Supplement). Between March 27 and May 6, 2020, we collected and analyzed 64 serial breastmilk samples from 18 SARS-CoV-2-infected women residing in the U.S. (see Supplement for clinical characteristics). Breastmilk samples were collected before and after women had a positive SARS-CoV-2 RT-PCR test and all but one woman had symptomatic disease (see Figure). One of the 64 breastmilk samples had detectable SARS-CoV-2 RNA by RT-PCR. The positive sample was collected on the day of symptom onset but one sample 2 days prior to symptom onset and two subsequent samples, collected 12 and 41 days later, tested negative for viral RNA. In addition, a subset of 26 breastmilk samples from nine women were tested for the presence of replication-competent virus using our established culture methods, and all were negative including the one sample that tested positive for viral RNA by RT-PCR. Although SARS-CoV-2 RNA was detected in one milk sample from one of eighteen infected women, the viral culture for that sample was negative. This suggests that SARS-CoV-2 RNA does not represent replication-competent virus and that breastmilk itself is likely not a source of infection for the infant. Furthermore, when control breastmilk samples spiked with replication-competent SARS-CoV-2 virus were treated by Holder pasteurization, a process commonly performed by donor milk banks, no replication-competent virus nor viral RNA was detectable. Further research to confirm these findings is needed, as well as an examination of convalescent milk for the presence of antibodies against SARS-CoV-2.