Extracorporeal Membrane Oxygenation-Dependent Fulminant Melioidosis From Caspase 4 Mutation Reversed by Interferon Gamma Therapy.

We describe bedside-to-bench immunological and genetic elucidation of defective pyroptosis attributable to novel caspase 4 defect mediating pathogen-triggered inflammatory programmed cell death, in the setting of severe pneumonia and abscess-forming melioidosis in an overtly healthy host failing to clear Burkholderia pseudomallei infection, and how targeted adjunctive biological therapy led to a successful outcome.

Viral Dynamics and Immune Correlates of Coronavirus Disease 2019 (COVID-19) Severity.

BACKGROUND: Key knowledge gaps remain in the understanding of viral dynamics and immune response of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection. METHODS: We evaluated these characteristics and established their association with clinical severity in a prospective observational cohort study of 100 patients with PCR-confirmed SARS-CoV-2 infection (mean age, 46 years; 56% male; 38% with comorbidities). Respiratory samples (n = 74) were collected for viral culture, serum samples for measurement of IgM/IgG levels (n = 30), and plasma samples for levels of inflammatory cytokines and chemokines (n = 81). Disease severity was correlated with results from viral culture, serologic testing, and immune markers. RESULTS: Fifty-seven (57%) patients developed viral pneumonia, of whom 20 (20%) required supplemental oxygen, including 12 (12%) with invasive mechanical ventilation. Viral culture from respiratory samples was positive for 19 of 74 patients (26%). No virus was isolated when the PCR cycle threshold (Ct) value was >30 or >14 days after symptom onset. Seroconversion occurred at a median (IQR) of 12.5 (9-18) days for IgM and 15.0 (12-20) days for IgG; 54/62 patients (87.1%) sampled at day 14 or later seroconverted. Severe infections were associated with earlier seroconversion and higher peak IgM and IgG levels. Levels of IP-10, HGF, IL-6, MCP-1, MIP-1α, IL-12p70, IL-18, VEGF-A, PDGF-BB, and IL-1RA significantly correlated with disease severity. CONCLUSIONS: We found virus viability was associated with lower PCR Ct value in early illness. A stronger antibody response was associated with disease severity. The overactive proinflammatory immune signatures offer targets for host-directed immunotherapy, which should be evaluated in randomized controlled trials.

Effects of a major deletion in the SARS-CoV-2 genome on the severity of infection and the inflammatory response: an observational cohort study.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with a 382-nucleotide deletion (∆382) in the open reading frame 8 (ORF8) region of the genome have been detected in Singapore and other countries. We investigated the effect of this deletion on the clinical features of infection. METHODS: We retrospectively identified patients who had been screened for the ∆382 variant and recruited to the PROTECT study-a prospective observational cohort study conducted at seven public hospitals in Singapore. We collected clinical, laboratory, and radiological data from patients' electronic medical records and serial blood and respiratory samples taken during hospitalisation and after discharge. Individuals infected with the ∆382 variant were compared with those infected with wild-type SARS-CoV-2. Exact logistic regression was used to examine the association between the infection groups and the development of hypoxia requiring supplemental oxygen (an indicator of severe COVID-19, the primary endpoint). Follow-up for the study's primary endpoint is completed. FINDINGS: Between Jan 22 and March 21, 2020, 278 patients with PCR-confirmed SARS-CoV-2 infection were screened for the ∆382 deletion and 131 were enrolled onto the study, of whom 92 (70%) were infected with the wild-type virus, ten (8%) had a mix of wild-type and ∆382-variant viruses, and 29 (22%) had only the ∆382 variant. Development of hypoxia requiring supplemental oxygen was less frequent in the ∆382 variant group (0 [0%] of 29 patients) than in the wild-type only group (26 [28%] of 92; absolute difference 28% [95% CI 14-28]). After adjusting for age and presence of comorbidities, infection with the ∆382 variant only was associated with lower odds of developing hypoxia requiring supplemental oxygen (adjusted odds ratio 0·07 [95% CI 0·00-0·48]) compared with infection with wild-type virus only. INTERPRETATION: The ∆382 variant of SARS-CoV-2 seems to be associated with a milder infection. The observed clinical effects of deletions in ORF8 could have implications for the development of treatments and vaccines. FUNDING: National Medical Research Council Singapore.

Update on Non-Culture-Based Diagnostics for Invasive Fungal Disease.

Diagnostic tests for fungi provide the mycological evidence to strengthen diagnosis of invasive fungal disease. Conventional microbiology and histopathology have their limitations. Recognizing this, there have been attempts at developing new methods to improve yield of diagnosing invasive fungal disease (IFD). The recent focus has been on non-culture-based antigen detection and molecular methods. The use of antigen detection of IFD through 1,3-ß-D-glucan and galactomannan assay have been expanded, followed by development of lateral flow assays, and in combination with other diagnostic modalities to further increase diagnostic yield. The molecular diagnostic front has seen initiatives to standardize polymerase chain reaction methodologies to detect fungi and anti-fungal resistance, new platforms such as the T2Candida Biosystems and foray into fungal metagenomics. As these newer assays undergo stringent validation before incorporation into the diagnostic algorithm, the clinician needs to be mindful of their bedside utility as well as their limitation.

The Divergent Immunomodulatory Effects of Short Chain Fatty Acids and Medium Chain Fatty Acids.

Fatty acids are derived from diet and fermentative processes by the intestinal flora. Two to five carbon chain fatty acids, termed short chain fatty acids (SCFA) are increasingly recognized to play a role in intestinal homeostasis. However, the characteristics of slightly longer 6 to 10 carbon, medium chain fatty acids (MCFA), derived primarily from diet, are less understood. Here, we demonstrated that SCFA and MCFA have divergent immunomodulatory propensities. SCFA down-attenuated host pro-inflammatory IL-1ß, IL-6, and TNFα response predominantly through the TLR4 pathway, whereas MCFA augmented inflammation through TLR2. Butyric (C4) and decanoic (C10) acid displayed most potent modulatory effects within the SCFA and MCFA, respectively. Reduction in TRAF3, IRF3 and TRAF6 expression were observed with butyric acid. Decanoic acid induced up-regulation of GPR84 and PPARγ and altered HIF-1α/HIF-2α ratio. These variant immune characteristics of the fatty acids which differ by just several carbon atoms may be attributable to their origins, with SCFA being primarily endogenous and playing a physiological role, and MCFA exogenously from the diet.

Effect of Remdesivir vs Standard Care on Clinical Status at 11 Days in Patients With Moderate COVID-19: A Randomized Clinical Trial.

Importance: Remdesivir demonstrated clinical benefit in a placebo-controlled trial in patients with severe coronavirus disease 2019 (COVID-19), but its effect in patients with moderate disease is unknown. Objective: To determine the efficacy of 5 or 10 days of remdesivir treatment compared with standard care on clinical status on day 11 after initiation of treatment. Design, Setting, and Participants: Randomized, open-label trial of hospitalized patients with confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and moderate COVID-19 pneumonia (pulmonary infiltrates and room-air oxygen saturation >94%) enrolled from March 15 through April 18, 2020, at 105 hospitals in the United States, Europe, and Asia. The date of final follow-up was May 20, 2020. Interventions: Patients were randomized in a 1:1:1 ratio to receive a 10-day course of remdesivir (n = 197), a 5-day course of remdesivir (n = 199), or standard care (n = 200). Remdesivir was dosed intravenously at 200 mg on day 1 followed by 100 mg/d. Main Outcomes and Measures: The primary end point was clinical status on day 11 on a 7-point ordinal scale ranging from death (category 1) to discharged (category 7). Differences between remdesivir treatment groups and standard care were calculated using proportional odds models and expressed as odds ratios. An odds ratio greater than 1 indicates difference in clinical status distribution toward category 7 for the remdesivir group vs the standard care group. Results: Among 596 patients who were randomized, 584 began the study and received remdesivir or continued standard care (median age, 57 [interquartile range, 46-66] years; 227 [39%] women; 56% had cardiovascular disease, 42% hypertension, and 40% diabetes), and 533 (91%) completed the trial. Median length of treatment was 5 days for patients in the 5-day remdesivir group and 6 days for patients in the 10-day remdesivir group. On day 11, patients in the 5-day remdesivir group had statistically significantly higher odds of a better clinical status distribution than those receiving standard care (odds ratio, 1.65; 95% CI, 1.09-2.48; P = .02). The clinical status distribution on day 11 between the 10-day remdesivir and standard care groups was not significantly different (P = .18 by Wilcoxon rank sum test). By day 28, 9 patients had died: 2 (1%) in the 5-day remdesivir group, 3 (2%) in the 10-day remdesivir group, and 4 (2%) in the standard care group. Nausea (10% vs 3%), hypokalemia (6% vs 2%), and headache (5% vs 3%) were more frequent among remdesivir-treated patients compared with standard care. Conclusions and Relevance: Among patients with moderate COVID-19, those randomized to a 10-day course of remdesivir did not have a statistically significant difference in clinical status compared with standard care at 11 days after initiation of treatment. Patients randomized to a 5-day course of remdesivir had a statistically significant difference in clinical status compared with standard care, but the difference was of uncertain clinical importance. Trial Registration: ClinicalTrials.gov Identifier: NCT04292730.

Genetic, immunological, and clinical features of patients with bacterial and fungal infections due to inherited IL-17RA deficiency.

Chronic mucocutaneous candidiasis (CMC) is defined as recurrent or persistent infection of the skin, nails, and/or mucosae with commensal Candida species. The first genetic etiology of isolated CMC-autosomal recessive (AR) IL-17 receptor A (IL-17RA) deficiency-was reported in 2011, in a single patient. We report here 21 patients with complete AR IL-17RA deficiency, including this first patient. Each patient is homozygous for 1 of 12 different IL-17RA alleles, 8 of which create a premature stop codon upstream from the transmembrane domain and have been predicted and/or shown to prevent expression of the receptor on the surface of circulating leukocytes and dermal fibroblasts. Three other mutant alleles create a premature stop codon downstream from the transmembrane domain, one of which encodes a surface-expressed receptor. Finally, the only known missense allele (p.D387N) also encodes a surface-expressed receptor. All of the alleles tested abolish cellular responses to IL-17A and -17F homodimers and heterodimers in fibroblasts and to IL-17E/IL-25 in leukocytes. The patients are currently aged from 2 to 35 y and originate from 12 unrelated kindreds. All had their first CMC episode by 6 mo of age. Fourteen patients presented various forms of staphylococcal skin disease. Eight were also prone to various bacterial infections of the respiratory tract. Human IL-17RA is, thus, essential for mucocutaneous immunity to Candida and Staphylococcus, but otherwise largely redundant. A diagnosis of AR IL-17RA deficiency should be considered in children or adults with CMC, cutaneous staphylococcal disease, or both, even if IL-17RA is detected on the cell surface.

Melioidosis, Singapore, 2003-2014.

In contrast with northern Australia and Thailand, in Singapore the incidence of melioidosis and co-incidence of melioidosis and pneumonia have declined. Burkholderia pseudomallei deep abscesses increased 20.4% during 2003-2014. These trends could not be explained by the environmental and climatic factors conventionally ascribed to melioidosis.

Distinguishing Zika and Dengue Viruses through Simple Clinical Assessment, Singapore.

Dengue virus and Zika virus coexist in tropical regions in Asia where healthcare resources are limited; differentiating the 2 viruses is challenging. We showed in a case-control discovery cohort, and replicated in a validation cohort, that the diagnostic indices of conjunctivitis, platelet count, and monocyte count reliably distinguished between these viruses.

The Fungal Mycobiome and Its Interaction with Gut Bacteria in the Host.

The advent of sequencing technology has endowed us with the capacity to study microbes constituting the human commensal community that were previously non-culturable. Much of the initial works have concentrated on the bacterial flora constituting the gut microbiome, since specimens are readily accessible in health and disease. Less, however, is understood of the "silent population"-the fungal species, also known as the mycobiome. Living in symbiosis with bacteria as commensals in our body, it is perceivable that the mycobiome exerts an inadvertent influence on the microbiome. We review here the recent knowledge gained from study of the interaction between the mycobiome and microbiome in health and disease susceptibility, immunity, and consequences from antimicrobial treatment.

Network Meta-analysis and Pharmacoeconomic Evaluation of Fluconazole, Itraconazole, Posaconazole, and Voriconazole in Invasive Fungal Infection Prophylaxis.

Invasive fungal infections (IFIs) are associated with high mortality rates and large economic burdens. Triazole prophylaxis is used for at-risk patients with hematological malignancies or stem cell transplants. We evaluated both the efficacy and the cost-effectiveness of triazole prophylaxis. A network meta-analysis (NMA) of randomized controlled trials (RCTs) evaluating fluconazole, itraconazole capsule and solution, posaconazole, and voriconazole was conducted. The outcomes of interest included the incidences of IFIs and deaths. This was coupled with a cost-effectiveness analysis from patient perspective over a lifetime horizon. Probabilities of transitions between health states were derived from the NMA. Resource use and costs were obtained from the Singapore health care institution. Data on 5,505 participants in 21 RCTs were included. Other than itraconazole capsule, all triazole antifungals were effective in reducing IFIs. Posaconazole was better than fluconazole (odds ratio [OR], 0.35 [95% confidence interval [CI], 0.16 to 0.73]) and itraconazole capsule (OR, 0.25 [95% CI, 0.06 to 0.97]), but not voriconazole (OR, 1.31 [95% CI, 0.43 to 4.01]), in preventing IFIs. Posaconazole significantly reduced all-cause deaths, compared to placebo, fluconazole, and itraconazole solution (OR, 0.49 to 0.54 [95% CI, 0.28 to 0.88]). The incremental cost-effectiveness ratio for itraconazole solution was lower than that for posaconazole (Singapore dollars [SGD] 12,546 versus SGD 26,817 per IFI avoided and SGD 5,844 versus SGD 12,423 per LY saved) for transplant patients. For leukemia patients, itraconazole solution was the dominant strategy. Voriconazole was dominated by posaconazole. All triazole antifungals except itraconazole capsule were effective in preventing IFIs. Posaconazole was more efficacious in reducing IFIs and all-cause deaths than were fluconazole and itraconazole. Both itraconazole solution and posaconazole were cost-effective in the Singapore health care setting.

Bimodal Influence of Vitamin D in Host Response to Systemic Candida Infection-Vitamin D Dose Matters.

Vitamin D level is linked to susceptibility to infections, but its relevance in candidemia is unknown. We aimed to investigate the in vivo sequelae of vitamin D3 supplementation in systemic Candida infection. Implicating the role of vitamin D in Candida infections, we showed that candidemic patients had significantly lower 25-OHD concentrations. Candida-infected mice treated with low-dose 1,25(OH)2D3 had reduced fungal burden and better survival relative to untreated mice. Conversely, higher 1,25(OH)2D3 doses led to poor outcomes. Mechanistically, low-dose 1,25(OH)2D3 induced proinflammatory immune responses. This was mediated through suppression of SOCS3 and induction of vitamin D receptor binding with the vitamin D-response elements in the promoter of the gene encoding interferon γ. These beneficial effects were negated with higher vitamin D3 doses. While the antiinflammatory effects of vitamin D3 are well described, we found that, conversely, lower doses conferred proinflammatory benefits in Candida infection. Our study highlights caution against extreme deviations of vitamin D levels during infections.

Association of melioidosis incidence with rainfall and humidity, Singapore, 2003-2012.

Soil has been considered the natural reservoir for the bacterium Burkholderia pseudomallei, which causes melioidosis. We examined 550 melioidosis cases that occurred during a 10-year period in the highly urbanized city of Singapore, where soil exposure is rare, and found that rainfall and humidity levels were associated with disease incidence.

Performance of Risk Scores in Predicting Infective Endocarditis in Patients with Staphylococcus aureus Bacteraemia in a Prospective Asian Cohort.

Background: Several risk scores have been derived to predict the risk of infective endocarditis (IE) amongst patients with Staphylococcus aureus bacteraemia (SAB), which helps to guide clinical management. Methods: We prospectively studied 634 patients admitted with SAB. The cohort was stratified into those with or without IE, and the PREDICT Day 1, Day 5 and VIRSTA scores were tabulated. Area under the receiver operating characteristic (AUC) curves were constructed to compare the performance of each score. Results: Of the 634 patients examined, 36 (5.7%) had IE. These patients were younger (51.6 ± 20.1 vs. 59.2 ± 18.0 years, p = 0.015), tended to have community acquisition of bacteraemia (41.7% vs. 17.9%, p < 0.001), and had persistent bacteraemia beyond 72 h (19.4% vs. 6.0%, p = 0.002). The VIRSTA score had the best performance in predicting IE (AUC 0.76, 95%CI 0.66-0.86) compared with PREDICT Day 1 and Day 5. A VIRSTA score of <3 had the best negative predictive value (97.5%), compared with PREDICT Day 1 (<4) and Day 5 (<2) (94.3% and 96.6%, respectively). Conclusions: Overall, the risk scores performed well in our Asian cohort. If applied, 23.5% of the cohort with a VIRSTA ≥ 3 would require TEE, and a score of <3 had an excellent negative predictive value.

Severe Invasive Infections Linked to Novel IRAK2 Immune Variants.

In subjects with peculiar susceptibility to severe infections by common pyogenic bacteria, mutations of interleukin-1 receptor-associated kinase proteins (IRAK)1 and IRAK4 had been identified. The IRAK kinases function as downstream signal transductors following activation of pathogen recognition receptors. In 2 patients with sequential or repeated invasive infections: herpes simplex virus-triggered hemophagocytic lymphohistiocytosis with tuberculosis, and Streptococcus pneumoniae bacteremia with candidemia respectively, novel mutations of IRAK2 were identified. These mutations compromised the capacity to ubiquinate (or functionally modify) the signal adaptor tumor necrosis factor receptor-associated factor 6 (TRAF6). The result is impairment of the cytokine TNF-alpha production. This susceptibility to a varied range of pathogens underlines a potential central role played by IRAK2 in mediating host defense in infectious diseases.

Adding Hyponatremia to the "Rule-of-6" Prediction Tool Improves Performance in Identifying Hospitalised Patients with COVID-19 at Risk of Adverse Clinical Outcomes.

The 'rule-of-6' prediction tool was shown to be able to identify COVID-19 patients at risk of adverse outcomes. During the pandemic, we frequently observed hyponatremia at presentation. We sought to evaluate if adding hyponatremia at presentation could improve the 'rule-of-6' prediction tool. We retrospectively analysed 1781 consecutive patients admitted to a single tertiary academic institution in Singapore with COVID-19 infection from February 2020 to October 2021. A total of 161 (9.0%) patients had hyponatremia. These patients were significantly older, with more co-morbidities and more likely to be admitted during the Delta wave (2021). They were more likely to have radiographic evidence of pneumonia (46.0% versus 13.0%, p < 0.001) and more adverse outcomes (25.5% vs. 4.1%, p < 0.001). Hyponatremia remained independently associated with adverse outcomes after adjusting for age, lack of medical co-morbidities, vaccination status, year of admission, CRP, LDH, and ferritin. The optimised cut-off for serum sodium in predicting adverse outcomes was approximately <135 mmol/L as determined by the Youden index. Although derived in early 2020, the 'rule-of-6' prediction tool continued to perform well in our later cohort (AUC: 0.72, 95%CI: 0.66-0.78). Adding hyponatremia to the 'rule-of-6' improved its performance (AUC: 0.76, 95%CI: 0.71-0.82). Patients with hyponatremia at presentation for COVID-19 had poorer outcomes even as new variants emerged.