Challenges and advances in the medical treatment of granulomatous amebic encephalitis.

Granulomatous amebic encephalitis, caused by the free-living amebae Balamuthia mandrillaris or Acanthamoeba species, is a rare and deadly infectious syndrome with a current mortality rate of >90%. Much work remains to define the optimal treatment for these infections. Here, we provide a comprehensive overview of the supporting evidence behind antimicrobials currently recommended by the Centers for Disease Control and Prevention (CDC) with updated statistics on survival rates and medication usage from the CDC Free-Living Ameba Database. We also discuss promising treatments, especially the emerging therapeutic agent nitroxoline, and provide recommendations for the next steps in this area.

In host evolution of beta lactam resistance during active treatment for Pseudomonas aeruginosa bacteremia.

Multidrug-resistant (MDR) Pseudomonas aeruginosa has been declared a serious threat by the United States Centers for Disease Control and Prevention. Here, we used whole genome sequencing (WGS) to investigate recurrent P. aeruginosa bloodstream infections in a severely immunocompromised patient. The infections demonstrated unusual, progressive increases in resistance to beta lactam antibiotics in the setting of active treatment with appropriate, guideline-directed agents. WGS followed by comparative genomic analysis of isolates collected over 44 days demonstrated in host evolution of a single P. aeruginosa isolate characterized by stepwise acquisition of two de-novo genetic resistance mechanisms over the course of treatment. We found a novel deletion affecting the ampC repressor ampD and neighboring gene ampE, which associated with initial cefepime treatment failure. This was followed by acquisition of a porin nonsense mutation, OprD, associated with resistance to carbapenems. This study highlights the potential for in-host evolution of P. aeruginosa during bloodstream infections in severely immunocompromised patients despite appropriate antimicrobial therapy. In addition, it demonstrates the utility of WGS for understanding unusual resistance patterns in the clinical context.

Clinical and biological heterogeneity of multisystem inflammatory syndrome in adults following SARS-CoV-2 infection: a case series.

Importance: Multisystem inflammatory syndrome in adults (MIS-A) is a poorly understood complication of SARS-CoV-2 infection with significant morbidity and mortality. Objective: Identify clinical, immunological, and histopathologic features of MIS-A to improve understanding of the pathophysiology and approach to treatment. Design: Three cases of MIS-A following SARS-CoV-2 infection were clinically identified between October 2021 - March 2022 using the U.S. Centers for Disease Control and Prevention diagnostic criteria. Clinical, laboratory, imaging, and tissue data were assessed. Findings: All three patients developed acute onset cardiogenic shock and demonstrated elevated inflammatory biomarkers at the time of hospital admission that resolved over time. One case co-occurred with new onset Type 1 diabetes and sepsis. Retrospective analysis of myocardial tissue from one case identified SARS-CoV-2 RNA. All three patients fully recovered with standard of care interventions plus immunomodulatory therapy that included intravenous immunoglobulin, corticosteroids, and in two cases, anakinra. Conclusion: MIS-A is a severe post-acute sequela of COVID-19 characterized by systemic elevation of inflammatory biomarkers. In this series of three cases, we find that although clinical courses and co-existent diseases vary, even severe presentations have potential for full recovery with prompt recognition and treatment. In addition to cardiogenic shock, glucose intolerance, unmasking of autoimmune disease, and sepsis can be features of MIS-A, and SARS-CoV-2 myocarditis can lead to a similar clinical syndrome.

Successful Treatment of Balamuthia mandrillaris Granulomatous Amebic Encephalitis with Nitroxoline.

A patient in California, USA, with rare and usually fatal Balamuthia mandrillaris granulomatous amebic encephalitis survived after receiving treatment with a regimen that included the repurposed drug nitroxoline. Nitroxoline, which is a quinolone typically used to treat urinary tract infections, was identified in a screen for drugs with amebicidal activity against Balamuthia.

Prevalence of type-1 interferon autoantibodies in adults with non-COVID-19 acute respiratory failure.

Auto-antibodies (Abs) to type I interferons (IFNs) are found in up to 25% of patients with severe COVID-19, and are implicated in disease pathogenesis. It has remained unknown, however, whether type I IFN auto-Abs are unique to COVID-19, or are also found in other types of severe respiratory illnesses. To address this, we studied a prospective cohort of 284 adults with acute respiratory failure due to causes other than COVID-19. We measured type I IFN auto-Abs by radio ligand binding assay and screened for respiratory viruses using clinical PCR and metagenomic sequencing. Three patients (1.1%) tested positive for type I IFN auto-Abs, and each had a different underlying clinical presentation. Of the 35 patients found to have viral infections, only one patient tested positive for type I IFN auto-Abs. Together, our data suggest that type I IFN auto-Abs are uncommon in critically ill patients with acute respiratory failure due to causes other than COVID-19.

Upper airway gene expression shows a more robust adaptive immune response to SARS-CoV-2 in children.

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults whereas disease burden in children is lower. To investigate whether differences in the upper airway immune response may contribute to this disparity, we compare nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 older adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes is robustly activated in both children and adults with SARS-CoV-2 infection compared to the respective non-viral groups, with only subtle distinctions. Children, however, demonstrate markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including response to TNF and production of IFNγ, IL-2 and IL-4. Cell type deconvolution confirms greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibit a decrease in proportions of ciliated cells, among the primary targets of SARS-CoV-2, upon infection. These findings demonstrate that children elicit a more robust innate and especially adaptive immune response to SARS-CoV-2 in the upper airway that likely contributes to their protection from severe disease in the lower airway.

Impact of e-cigarette aerosol on primary human alveolar epithelial type 2 cells.

Electronic cigarettes (e-cigarettes) are designed to simulate combustible cigarette smoking and to aid in smoking cessation. Although the number of e-cigarette users has been increasing, the potential health impacts and biological effects of e-cigarettes are still not fully understood. Previous research has focused on the biological effects of e-cigarettes on lung cancer cell lines and distal airway epithelial cells; however, there have been few published studies on the effect of e-cigarettes on primary lung alveolar epithelial cells. The primary purpose of this study was to investigate the direct effect of e-cigarette aerosol on primary human lung alveolar epithelial type 2 (AT2) cells, both alone and in the presence of viral infection. The Melo-3 atomizer caused direct AT2 cell toxicity, whereas the more popular Juul pod's aerosol did not have a detectable cytotoxic effect on AT2 cells. Juul nicotine aerosol also did not increase short-term susceptibility to viral infection. However, 3 days of exposure upregulated genes central to the generation of reactive oxygen species, lipid peroxidation, and carcinogen metabolism and downregulated key innate immune system genes related to cytokine and chemokine signaling. These findings have implications for the potentially injurious impact of long-term use of popular low-power e-cigarette pods on the human alveolar epithelium. Gene expression data might be an important endpoint for evaluating the potential harmful effects of vaping devices that do not cause overt toxicity.

Pneumonia surveillance with culture-independent metatranscriptomics in HIV-positive adults in Uganda: a cross-sectional study.

BACKGROUND: Pneumonia is a leading cause of death worldwide and is a major health-care challenge in people living with HIV. Despite this, the causes of pneumonia in this population remain poorly understood. We aimed to assess the feasibility of metatranscriptomics for epidemiological surveillance of pneumonia in patients with HIV in Uganda. METHODS: We performed a retrospective observational study in patients with HIV who were admitted to Mulago Hospital, Kampala, Uganda between Oct 1, 2009, and Dec 31, 2011. Inclusion criteria were age 18 years or older, HIV-positivity, and clinically diagnosed pneumonia. Exclusion criteria were contraindication to bronchoscopy or an existing diagnosis of tuberculosis. Bronchoalveolar lavage fluid was collected within 72 h of admission and a combination of RNA sequencing and Mycobacterium tuberculosis culture plus PCR were performed. The primary outcome was detection of an established or possible respiratory pathogen in the total study population. FINDINGS: We consecutively enrolled 217 patients during the study period. A potential microbial cause for pneumonia was identified in 211 (97%) patients. At least one microorganism of established respiratory pathogenicity was identified in 113 (52%) patients, and a microbe of possible pathogenicity was identified in an additional 98 (45%). M tuberculosis was the most commonly identified established pathogen (35 [16%] patients; in whom bacterial or viral co-infections were identified in 13 [37%]). Streptococcus mitis, although not previously reported as a cause of pneumonia in patients with HIV, was the most commonly identified bacterial organism (37 [17%] patients). Haemophilus influenzae was the most commonly identified established bacterial pathogen (20 [9%] patients). Pneumocystis jirovecii was only identified in patients with a CD4 count of less than 200 cells per mL. INTERPRETATION: We show the feasibility of using metatranscriptomics for epidemiologic surveillance of pneumonia by describing the spectrum of respiratory pathogens in adults with HIV in Uganda. Applying these methods to a contemporary cohort could enable broad assessment of changes in pneumonia aetiology following the emergence of SARS-CoV-2. FUNDING: US National Institutes of Health, Chan Zuckerberg Biohub.

Tracheal aspirate RNA sequencing identifies distinct immunological features of COVID-19 ARDS.

The immunological features that distinguish COVID-19-associated acute respiratory distress syndrome (ARDS) from other causes of ARDS are incompletely understood. Here, we report the results of comparative lower respiratory tract transcriptional profiling of tracheal aspirate from 52 critically ill patients with ARDS from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a "cytokine storm," we observe reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS is characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity. In silico analysis of gene expression identifies several candidate drugs that may modulate gene expression in COVID-19 ARDS, including dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 is characterized by impaired interferon-stimulated gene (ISG) expression. The relationship between SARS-CoV-2 viral load and expression of ISGs is decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients reveals distinct immunological features of COVID-19 ARDS.

Upper airway gene expression reveals a more robust innate and adaptive immune response to SARS-CoV-2 in children compared with older adults.

Unlike other respiratory viruses, SARS-CoV-2 disproportionately causes severe disease in older adults and only rarely in children. To investigate whether differences in the upper airway immune response could contribute to this disparity, we compared nasopharyngeal gene expression in 83 children (<19-years-old; 38 with SARS-CoV-2, 11 with other respiratory viruses, 34 with no virus) and 154 adults (>40-years-old; 45 with SARS-CoV-2, 28 with other respiratory viruses, 81 with no virus). Expression of interferon-stimulated genes (ISGs) was robustly activated in both children and adults with SARS-CoV-2 compared to the respective non-viral groups, with only relatively subtle distinctions. Children, however, demonstrated markedly greater upregulation of pathways related to B cell and T cell activation and proinflammatory cytokine signaling, including TNF, IFNγ, IL-2 and IL-4 production. Cell type deconvolution confirmed greater recruitment of B cells, and to a lesser degree macrophages, to the upper airway of children. Only children exhibited a decrease in proportions of ciliated cells, the primary target of SARS-CoV-2, upon infection with the virus. These findings demonstrate that children elicit a more robust innate and adaptive immune response to SARS-CoV-2 infection in the upper airway that likely contributes to their protection from severe disease in the lower airway.

COVID-19 ARDS is characterized by a dysregulated host response that differs from cytokine storm and is modified by dexamethasone.

We performed comparative lower respiratory tract transcriptional profiling of 52 critically ill patients with the acute respiratory distress syndrome (ARDS) from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a cytokine storm, we observed reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS was characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity that were predicted to be modulated by dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 was characterized by impaired interferon-stimulated gene expression (ISG). We found that the relationship between SARS-CoV-2 viral load and expression of ISGs was decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients with COVID-19 ARDS did not demonstrate cytokine storm but instead revealed a unique and dysregulated host response predicted to be modified by dexamethasone.

Upper airway gene expression reveals suppressed immune responses to SARS-CoV-2 compared with other respiratory viruses.

SARS-CoV-2 infection is characterized by peak viral load in the upper airway prior to or at the time of symptom onset, an unusual feature that has enabled widespread transmission of the virus and precipitated a global pandemic. How SARS-CoV-2 is able to achieve high titer in the absence of symptoms remains unclear. Here, we examine the upper airway host transcriptional response in patients with COVID-19 (n = 93), other viral (n = 41) or non-viral (n = 100) acute respiratory illnesses (ARIs). Compared with other viral ARIs, COVID-19 is characterized by a pronounced interferon response but attenuated activation of other innate immune pathways, including toll-like receptor, interleukin and chemokine signaling. The IL-1 and NLRP3 inflammasome pathways are markedly less responsive to SARS-CoV-2, commensurate with a signature of diminished neutrophil and macrophage recruitment. This pattern resembles previously described distinctions between symptomatic and asymptomatic viral infections and may partly explain the propensity for pre-symptomatic transmission in COVID-19. We further use machine learning to build 27-, 10- and 3-gene classifiers that differentiate COVID-19 from other ARIs with AUROCs of 0.981, 0.954 and 0.885, respectively. Classifier performance is stable across a wide range of viral load, suggesting utility in mitigating false positive or false negative results of direct SARS-CoV-2 tests.

Clinical features, diagnostics, and outcomes of patients presenting with acute respiratory illness: A retrospective cohort study of patients with and without COVID-19.

BACKGROUND: Most data on the clinical presentation, diagnostics, and outcomes of patients with COVID-19 have been presented as case series without comparison to patients with other acute respiratory illnesses. METHODS: We examined emergency department patients between February 3 and March 31, 2020 with an acute respiratory illness who were tested for SARS-CoV-2. We determined COVID-19 status by PCR and metagenomic next generation sequencing (mNGS). We compared clinical presentation, diagnostics, treatment, and outcomes. FINDINGS: Among 316 patients, 33 tested positive for SARS-CoV-2; 31 without COVID-19 tested positive for another respiratory virus. Among patients with additional viral testing (27/33), no SARS-CoV-2 co-infections were identified. Compared to those who tested negative, patients with COVID-19 reported longer symptoms duration (median 7d vs. 3d, p < 0.001). Patients with COVID-19 were more often hospitalized (79% vs. 56%, p = 0.014). When hospitalized, patients with COVID-19 had longer hospitalizations (median 10.7d vs. 4.7d, p < 0.001) and more often developed ARDS (23% vs. 3%, p < 0.001). Most comorbidities, medications, symptoms, vital signs, laboratories, treatments, and outcomes did not differ by COVID-19 status. INTERPRETATION: While we found differences in clinical features of COVID-19 compared to other acute respiratory illnesses, there was significant overlap in presentation and comorbidities. Patients with COVID-19 were more likely to be admitted to the hospital, have longer hospitalizations and develop ARDS, and were unlikely to have co-existent viral infections. FUNDING: National Center for Advancing Translational Sciences, National Heart Lung Blood Institute, National Institute of Allergy and Infectious Diseases, Chan Zuckerberg Biohub, Chan Zuckerberg Initiative.

Upper airway gene expression differentiates COVID-19 from other acute respiratory illnesses and reveals suppression of innate immune responses by SARS-CoV-2.

We studied the host transcriptional response to SARS-CoV-2 by performing metagenomic sequencing of upper airway samples in 238 patients with COVID-19, other viral or non-viral acute respiratory illnesses (ARIs). Compared to other viral ARIs, COVID-19 was characterized by a diminished innate immune response, with reduced expression of genes involved in toll-like receptor and interleukin signaling, chemokine binding, neutrophil degranulation and interactions with lymphoid cells. Patients with COVID-19 also exhibited significantly reduced proportions of neutrophils and macrophages, and increased proportions of goblet, dendritic and B-cells, compared to other viral ARIs. Using machine learning, we built 26-, 10- and 3-gene classifiers that differentiated COVID-19 from other acute respiratory illnesses with AUCs of 0.980, 0.950 and 0.871, respectively. Classifier performance was stable at low viral loads, suggesting utility in settings where direct detection of viral nucleic acid may be unsuccessful. Taken together, our results illuminate unique aspects of the host transcriptional response to SARS-CoV-2 in comparison to other respiratory viruses and demonstrate the feasibility of COVID-19 diagnostics based on patient gene expression.

Clinical features, diagnostics, and outcomes of patients presenting with acute respiratory illness: a comparison of patients with and without COVID-19.

BACKGROUND: Emerging data on the clinical presentation, diagnostics, and outcomes of patients with COVID-19 have largely been presented as case series. Few studies have compared these clinical features and outcomes of COVID-19 to other acute respiratory illnesses. METHODS: We examined all patients presenting to an emergency department in San Francisco, California between February 3 and March 31, 2020 with an acute respiratory illness who were tested for SARS-CoV-2. We determined COVID-19 status by PCR and metagenomic next generation sequencing (mNGS). We compared demographics, comorbidities, symptoms, vital signs, and laboratory results including viral diagnostics using PCR and mNGS. Among those hospitalized, we determined differences in treatment (antibiotics, antivirals, respiratory support) and outcomes (ICU admission, ICU interventions, acute respiratory distress syndrome, cardiac injury). FINDINGS: In a cohort of 316 patients, 33 (10%) tested positive for SARS-CoV-2; 31 patients, all without COVID-19, tested positive for another respiratory virus (16%). Among patients with additional viral testing, no co-infections with SARS-CoV-2 were identified by PCR or mNGS. Patients with COVID-19 reported longer symptoms duration (median 7 vs. 3 days), and were more likely to report fever (82% vs. 44%), fatigue (85% vs. 50%), and myalgias (61% vs 27%); p<0.001 for all comparisons. Lymphopenia (55% vs 34%, p=0.018) and bilateral opacities on initial chest radiograph (55% vs. 24%, p=0.001) were more common in patients with COVID-19. Patients with COVID-19 were more often hospitalized (79% vs. 56%, p=0.014). Of 186 hospitalized patients, patients with COVID-19 had longer hospitalizations (median 10.7d vs. 4.7d, p<0.001) and were more likely to develop ARDS (23% vs. 3%, p<0.001). Most comorbidities, home medications, signs and symptoms, vital signs, laboratory results, treatment, and outcomes did not differ by COVID-19 status. INTERPRETATION: While we found differences in clinical features of COVID-19 compared to other acute respiratory illnesses, there was significant overlap in presentation and comorbidities. Patients with COVID-19 were more likely to be admitted to the hospital, have longer hospitalizations and develop ARDS, and were unlikely to have co-existent viral infections. These findings enhance understanding of the clinical characteristics of COVID-19 in comparison to other acute respiratory illnesses. .

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity.

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

ANALYSIS OF PLASMODIUM LINEAGES IDENTIFIED IN CAPTIVE PENGUINS (SPHENISCIFORMES SPP.), EIDERS (SOMATERIA SPP.), AND INCA TERNS (LAROSTERNA INCA) IN A NORTH AMERICAN ZOOLOGICAL COLLECTION.

Vector-borne Plasmodium spp. infect a wide range of bird species. Although infections may be asymptomatic, certain genera, especially those that evolved in regions without endemic malaria, appear particularly susceptible to symptomatic disease, leading to morbidity and mortality. High mortalities associated with malaria infections have been documented in captive species of Sphenisciformes, Somateria, and Larosterna, all genera that evolved in climates with low mosquito exposure. To better characterize trends in Plasmodium-related mortality in a zoological collection in New York, necropsy reports for birds of all three genera that died between 1998 and February 2018 were analyzed; comparisons were made between birds that died with or without evidence of malaria infection. A seasonal peak in deaths was observed in birds regardless of their malaria status. There was no significant difference in the age of birds at death between malaria-positive and malaria-negative animals. These results suggest that age and season of death were not associated with malaria status. To investigate an association between parasite lineage and clinical outcome, polymerase chain reaction was used to identify parasite lineage in necropsied birds as well as healthy birds sampled as part of surveillance studies. Twelve different Plasmodium lineages were identified. The relative prevalence of parasite lineages was compared between necropsy and surveillance samples. A single parasite lineage, SGS1 (species: Plasmodium relictum), was significantly more likely to be found in surveillance samples; it was detected in a plurality of surveillance data but found in only one necropsy case. Other parasite lineages were more likely to be found in necropsies than in surveillance samples, most notably SEIAUR01 (species: Plasmodium cathemerium). These data may be consistent with a difference in virulence between parasite lineages. This investigation has implications for the monitoring and care of vulnerable avian species.

Nontyphoidal Salmonella as a Cause of Mediastinal Abscess after Aortic Valve Replacement: A Case Report and Review of Postoperative Salmonella Infections.

BACKGROUND: Nontyphoidal Salmonella (NTS) is a pathogen that causes several human clinical illnesses, most commonly gastroenteritis. Focal infections are rare and are generally reported in the gastrointestinal and genitourinary systems. Very few studies have reported NTS abscess as a postoperative complication. CASE REPORT: We describe an elderly patient who developed NTS bacteremia and mediastinal abscess after aortic valve replacement. CONCLUSIONS: This report describes an extremely rare occurrence of an NTS mediastinal abscess complicating a surgical procedure. The patient may have acquired the pathogen from a snake kept as a family pet and likely developed bacteremia followed by seeding of the surgically damaged tissues.