The global role of G6PD in infection and immunity.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans. G6PD is an essential enzyme in the pentose phosphate pathway (PPP), generating NADPH needed for cellular biosynthesis and reactive oxygen species (ROS) homeostasis, the latter especially key in red blood cells (RBCs). Beyond the RBC, there is emerging evidence that G6PD exerts an immunologic role by virtue of its functions in leukocyte oxidative metabolism and anabolic synthesis necessary for immune effector function. We review these here, and consider the global immunometabolic role of G6PD activity and G6PD deficiency in modulating inflammation and immunopathology.

Surgical and Medical Management of Purulent Bacterial Pericarditis With Pericardial Mass in a Young Child.

Purulent bacterial pericarditis is rare and associated with significant short- and long-term morbidity. We report a case of purulent bacterial pericarditis caused by Group A Streptococcus in an immunocompetent young child presenting with a pericardial mass. She was successfully treated with a combined medical and early surgical approach. (Level of Difficulty: Intermediate.).

Low Diagnostic Utility of Frequent Serial Tracheal Aspirate Cultures in the PICU.

OBJECTIVES: To determine the diagnostic outcomes of serial tracheal aspirate cultures (TACs) in the PICU. DESIGN: A retrospective chart review of TAC utilization was performed. Items recorded for each TAC included the time and date of culture acquisition, result, changes in microbial resistance patterns, antimicrobial therapy, and patient clinical course. SETTING: A single urban tertiary care children's hospital in the United States. SUBJECTS: Patients admitted to the PICU from January 1, to October 31, 2021, for whom a TAC was performed. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: One hundred fifty unique subjects had 582 TACs performed during the study period, of which 145 (24.9%) were serially repeated within 72 hours. Of these serial TACs, 82 (56.6%) had no growth, 41 (28.3%) grew the same organism as the prior culture, with most (36/41) displaying no major change in antimicrobial susceptibilities, 11 (7.6%) grew a new organism previously grown during the admission, and 11 (7.6%) grew a new organism not previously grown during the admission. Overall, only 26 of these serial TACs (17.9%) provided new diagnostic information, whereas only five (3.4%) led to a change in management. CONCLUSIONS: Frequent serial TAC sampling in the PICU is common and infrequently yields new data that impact clinical decision-making. Considering worsening antimicrobial resistance and the role of diagnostic stewardship in mitigating it, these findings further support a 72-hour reassessment period before performing a repeat TAC in critically ill children.

HIV infection drives pro-inflammatory immunothrombotic pathway activation and organ dysfunction among adults with sepsis in Uganda.

BACKGROUND: The global burden of sepsis is concentrated in high HIV-burden settings in sub-Saharan Africa (SSA). Despite this, little is known about the immunopathology of sepsis in persons with HIV (PWH) in the region. We sought to determine the influence of HIV on host immune responses and organ dysfunction among adults hospitalized with suspected sepsis in Uganda. DESIGN: Prospective cohort study. METHODS: We compared organ dysfunction and 30-day outcome profiles of PWH and those without HIV. We quantified 14 soluble immune mediators, reflective of key domains of sepsis immunopathology, and performed whole-blood RNA-sequencing on samples from a subset of patients. We used propensity score methods to match PWH and those without HIV by demographics, illness duration, and clinical severity, and compared immune mediator concentrations and gene expression profiles across propensity score-matched groups. RESULTS: Among 299 patients, 157 (52.5%) were PWH (clinical stage 3 or 4 in 80.3%, 67.7% with known HIV on antiretroviral therapy). PWH presented with more severe physiologic derangement and shock, and had higher 30-day mortality (34.5% vs. 10.2%; P  < 0.001). Across propensity score-matched groups, PWH exhibited greater pro-inflammatory immune activation, including upregulation of interleukin (IL)-6, IL-8, IL-15, IL-17 and HMGB1 signaling, with concomitant T-cell exhaustion, prothrombotic pathway activation, and angiopoeitin-2-related endothelial dysfunction. CONCLUSIONS: Sepsis-related organ dysfunction and mortality in Uganda disproportionately affect PWH, who demonstrate exaggerated activation of multiple immunothrombotic and metabolic pathways implicated in sepsis pathogenesis. Further investigations are needed to refine understanding of sepsis immunopathology in PWH, particularly mechanisms amenable to therapeutic manipulation.

Klebsiella pneumoniae induces host metabolic stress that promotes tolerance to pulmonary infection.

K. pneumoniae sequence type 258 (Kp ST258) is a major cause of healthcare-associated pneumonia. However, it remains unclear how it causes protracted courses of infection in spite of its expression of immunostimulatory lipopolysaccharide, which should activate a brisk inflammatory response and bacterial clearance. We predicted that the metabolic stress induced by the bacteria in the host cells shapes an immune response that tolerates infection. We combined in situ metabolic imaging and transcriptional analyses to demonstrate that Kp ST258 activates host glutaminolysis and fatty acid oxidation. This response creates an oxidant-rich microenvironment conducive to the accumulation of anti-inflammatory myeloid cells. In this setting, metabolically active Kp ST258 elicits a disease-tolerant immune response. The bacteria, in turn, adapt to airway oxidants by upregulating the type VI secretion system, which is highly conserved across ST258 strains worldwide. Thus, much of the global success of Kp ST258 in hospital settings can be explained by the metabolic activity provoked in the host that promotes disease tolerance.

Multidimensional analysis of the host response reveals prognostic and pathogen-driven immune subtypes among adults with sepsis in Uganda.

BACKGROUND: The global burden of sepsis is concentrated in sub-Saharan Africa, where severe infections disproportionately affect young, HIV-infected adults and high-burden pathogens are unique. In this context, poor understanding of sepsis immunopathology represents a crucial barrier to development of locally-effective treatment strategies. We sought to determine inter-individual immunologic heterogeneity among adults hospitalized with sepsis in a sub-Saharan African setting, and characterize associations between immune subtypes, infecting pathogens, and clinical outcomes. METHODS: Among a prospective observational cohort of 288 adults hospitalized with suspected sepsis in Uganda, we applied machine learning methods to 14 soluble host immune mediators, reflective of key domains of sepsis immunopathology (innate and adaptive immune activation, endothelial dysfunction, fibrinolysis), to identify immune subtypes in randomly-split discovery (N = 201) and internal validation (N = 87) sub-cohorts. In parallel, we applied similar methods to whole-blood RNA-sequencing data from a consecutive subset of patients (N = 128) to identify transcriptional subtypes, which we characterized using biological pathway and immune cell-type deconvolution analyses. RESULTS: Unsupervised clustering consistently identified two immune subtypes defined by differential activation of pro-inflammatory innate and adaptive immune pathways, with transcriptional evidence of concomitant CD56(-)/CD16( +) NK-cell expansion, T-cell exhaustion, and oxidative-stress and hypoxia-induced metabolic and cell-cycle reprogramming in the hyperinflammatory subtype. Immune subtypes defined by greater pro-inflammatory immune activation, T-cell exhaustion, and metabolic reprogramming were consistently associated with a high-prevalence of severe and often disseminated HIV-associated tuberculosis, as well as more extensive organ dysfunction, worse functional outcomes, and higher 30-day mortality. CONCLUSIONS: Our results highlight unique host- and pathogen-driven features of sepsis immunopathology in sub-Saharan Africa, including the importance of severe HIV-associated tuberculosis, and reinforce the need to develop more biologically-informed treatment strategies in the region, particularly those incorporating immunomodulation.

An acquired acyltransferase promotes Klebsiella pneumoniae ST258 respiratory infection.

Klebsiella pneumoniae ST258 is a human pathogen associated with poor outcomes worldwide. We identify a member of the acyltransferase superfamily 3 (atf3), enriched within the ST258 clade, that provides a major competitive advantage for the proliferation of these organisms in vivo. Comparison of a wild-type ST258 strain (KP35) and a Δatf3 isogenic mutant generated by CRISPR-Cas9 targeting reveals greater NADH:ubiquinone oxidoreductase transcription and ATP generation, fueled by increased glycolysis. The acquisition of atf3 induces changes in the bacterial acetylome, promoting lysine acetylation of multiple proteins involved in central metabolism, specifically Zwf (glucose-6 phosphate dehydrogenase). The atf3-mediated metabolic boost leads to greater consumption of glucose in the host airway and increased bacterial burden in the lung, independent of cytokine levels and immune cell recruitment. Acquisition of this acyltransferase enhances fitness of a K. pneumoniae ST258 isolate and may contribute to the success of this clonal complex as a healthcare-associated pathogen.

What about tocilizumab? A retrospective study from a NYC Hospital during the COVID-19 outbreak.

BACKGROUND: Tocilizumab, an interleukin-6 receptor blocker, has been used in the inflammatory phase of COVID-19, but its impact independent of corticosteroids remains unclear in patients with severe disease. METHODS: In this retrospective analysis of patients with COVID-19 admitted between March 2 and April 14, 2020 to a large academic medical center in New York City, we describe outcomes associated with tocilizumab 400 mg (without methylprednisolone) compared to a propensity-matched control. The primary endpoints were change in a 7-point ordinal scale of oxygenation and ventilator free survival, both at days 14 and 28. Secondary endpoints include incidence of bacterial superinfections and gastrointestinal perforation. Primary outcomes were evaluated using t-test. RESULTS: We identified 33 patients who received tocilizumab and matched 74 controls based on demographics and health measures upon admission. After adjusting for illness severity and baseline ordinal scale, we failed to find evidence of an improvement in hypoxemia based on an ordinal scale at hospital day 14 in the tocilizumab group (OR 2.2; 95% CI, 0.7-6.5; p = 0.157) or day 28 (OR 1.1; 95% CI, 0.4-3.6; p = 0.82). There also was no evidence of an improvement in ventilator-free survival at day 14 (OR 0.8; 95% CI, 0.18-3.5; p = 0.75) or day 28 (OR 1.1; 95% CI, 0.1-1.8; p = 0.23). There was no increase in secondary bacterial infection rates in the tocilizumab group compared to controls (OR 0.37; 95% CI, 0.09-1.53; p = 0.168). CONCLUSIONS: There was no evidence to support an improvement in hypoxemia or ventilator-free survival with use of tocilizumab 400 mg in the absence of corticosteroids. No increase in secondary bacterial infections was observed in the group receiving tocilizumab.

Hemozoin Promotes Lung Inflammation via Host Epithelial Activation.

Respiratory distress in severe malaria is associated with high mortality, but its pathogenesis remains unclear. The malaria pigment hemozoin (HZ) is abundant in target organs of severe malaria, including the lungs, and is known to be a potent innate immune activator of phagocytes. We hypothesized that HZ might also stimulate lung epithelial activation and thereby potentiate lung inflammation. We show here that airway epithelium stimulated with HZ undergoes global transcriptional reprogramming and changes in cell surface protein expression that comprise an epithelial activation phenotype. Proinflammatory signaling is induced, and key cytoadherence molecules are upregulated, including several associated with severe malaria, such as CD36 and ICAM1. Epithelial and extracellular matrix remodeling pathways are transformed, including induction of key metalloproteases and modulation of epithelial junctions. The overall program induced by HZ serves to promote inflammation and neutrophil transmigration, and is recapitulated in a murine model of HZ-induced acute pneumonitis. Together, our data demonstrate a direct role for hemozoin in stimulating epithelial activation that could potentiate lung inflammation in malaria.IMPORTANCE Respiratory distress (RD) is a complication of severe malaria associated with a particularly high risk for death in African children infected with the parasite Plasmodium falciparum The pathophysiology underlying RD remains poorly understood, and the condition is managed supportively. The parasite-derived factor HZ accumulates in target organs of severe malaria, including the lungs, and is a potent stimulator of immune cells. Our findings demonstrate that HZ causes global activation of lung epithelial cells, a response that directly promotes lung inflammation. HZ stimulates expression of key proinflammatory and cell surface molecules, alters signaling pathways involved in epithelial-matrix remodeling, and promotes neutrophil transmigration and airway inflammation. The lung epithelial activation induced by HZ mimics patterns seen in malarial lung injury and provides new insights into the molecular pathogenesis of RD.

Heterogeneous alleles comprising G6PD deficiency trait in West Africa exert contrasting effects on two major clinical presentations of severe malaria.

BACKGROUND: Glucose-6-phosphate dehydrogenase (G6PD) deficiency exhibits considerable allelic heterogeneity which manifests with variable biochemical and clinical penetrance. It has long been thought that G6PD deficiency confers partial protection against severe malaria, however prior genetic association studies have disagreed with regard to the strength and specificity of a protective effect, which might reflect differences in the host genetic background, environmental influences, or in the specific clinical phenotypes considered. METHODS: A case-control association study of severe malaria was conducted in The Gambia, a region in West Africa where there is considerable allelic heterogeneity underlying expression of G6PD deficiency trait, evaluating the three major nonsynonymous polymorphisms known to be associated with enzyme deficiency (A968G, T542A, and C202T) in a cohort of 3836 controls and 2379 severe malaria cases. RESULTS: Each deficiency allele exhibited a similar trend toward protection against severe malaria overall (15-26% reduced risk); however, in stratifying severe malaria to two of its constituent clinical subphenotypes, severe malarial anaemia (SMA) and cerebral malaria (CM), the three deficiency alleles exhibited trends of opposing effect, with risk conferred to SMA and protection with respect to CM. To assess the overall effect of G6PD deficiency trait, deficiency alleles found across all three loci were pooled. G6PD deficiency trait was found to be significantly associated with protection from severe malaria overall (OR 0.83 [0.75-0.92], P = 0.0006), but this was limited to CM (OR 0.73 [0.61-0.87], P = 0.0005), with a trend toward increased risk for SMA, especially in fully-deficient individuals (OR 1.43 [0.99-2.08], P = 0.056). Sex-stratified testing largely comported with these results, with evidence suggesting that protection by G6PD deficiency trait is conferred to both males and females, though susceptibility to SMA may be restricted to fully-deficient male hemizygotes. CONCLUSIONS: In a part of Africa where multiple alleles contribute to expression of G6PD deficiency trait, these findings clarify and extend previous work done in populations where a single variant predominates, and taken together suggest a causal role for G6PD deficiency trait itself with respect to severe malaria, with opposing effects seen on two major clinical subphenotypes.

Genetic determinants of glucose-6-phosphate dehydrogenase activity in Kenya.

BACKGROUND: The relationship between glucose-6-phosphate dehydrogenase (G6PD) deficiency and clinical phenomena such as primaquine-sensitivity and protection from severe malaria remains poorly defined, with past association studies yielding inconsistent and conflicting results. One possibility is that examination of a single genetic variant might underestimate the presence of true effects in the presence of unrecognized functional allelic diversity. METHODS: We systematically examined this possibility in Kenya, conducting a fine-mapping association study of erythrocyte G6PD activity in 1828 Kenyan children across 30 polymorphisms at or around the G6PD locus. RESULTS: We demonstrate a strong functional role for c.202G>A (rs1050828), which accounts for the majority of variance in enzyme activity observed (P=1.5×10⁻²°°, additive model). Additionally, we identify other common variants that exert smaller, intercorrelated effects independent of c.202G>A, and haplotype analyses suggest that each variant tags one of two haplotype motifs that are opposite in sequence identity and effect direction. We posit that these effects are of biological and possible clinical significance, specifically noting that c.376A>G (rs1050829) augments 202AG heterozygote risk for deficiency trait by two-fold (OR = 2.11 [1.12 - 3.84], P=0.014). CONCLUSIONS: Our results suggest that c.202G>A is responsible for the majority of the observed prevalence of G6PD deficiency trait in Kenya, but also identify a novel role for c.376A>G as a genetic modifier which marks a common haplotype that augments the risk conferred to 202AG heterozygotes, suggesting that variation at both loci merits consideration in genetic association studies probing G6PD deficiency-associated clinical phenotypes.

A novel cytofluorometric assay for the detection and quantification of glucose-6-phosphate dehydrogenase deficiency.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked enzymopathy that affects hundreds of millions of people worldwide, conferring increased risk of neonatal jaundice and oxidant-induced hemolytic anemia. Screening and diagnosis of G6PD deficiency is currently performed using genetic or biochemical assays, the former being cost ineffective in populations with significant allelic heterogeneity, and the latter being limited in ability to detect female heterozygotes. Cytochemical assays can obviate these shortcomings, but at the expense of added technical complexity and labor. We describe here a simple, novel cytofluorometric method that extends the classic methemoglobin reduction test, assessing G6PD deficiency at the level of an individual erythrocyte. In preliminary testing in Malian children, there was strong concordance between our method and established genetic and biochemical techniques. The assay is robust and economical, and could serve as a screening method as well as a research tool, especially for high-throughput applications such as flow cytometry.