IDentif.AI-Omicron: Harnessing an AI-Derived and Disease-Agnostic Platform to Pinpoint Combinatorial Therapies for Clinically Actionable Anti-SARS-CoV-2 Intervention.

Nanomedicine-based and unmodified drug interventions to address COVID-19 have evolved over the course of the pandemic as more information is gleaned and virus variants continue to emerge. For example, some early therapies (e.g., antibodies) have experienced markedly decreased efficacy. Due to a growing concern of future drug resistant variants, current drug development strategies are seeking to find effective drug combinations. In this study, we used IDentif.AI, an artificial intelligence-derived platform, to investigate the drug-drug and drug-dose interaction space of six promising experimental or currently deployed therapies at various concentrations: EIDD-1931, YH-53, nirmatrelvir, AT-511, favipiravir, and auranofin. The drugs were tested in vitro against a live B.1.1.529 (Omicron) virus first in monotherapy and then in 50 strategic combinations designed to interrogate the interaction space of 729 possible combinations. Key findings and interactions were then further explored and validated in an additional experimental round using an expanded concentration range. Overall, we found that few of the tested drugs showed moderate efficacy as monotherapies in the actionable concentration range, but combinatorial drug testing revealed significant dose-dependent drug-drug interactions, specifically between EIDD-1931 and YH-53, as well as nirmatrelvir and YH-53. Checkerboard validation analysis confirmed these synergistic interactions and also identified an interaction between EIDD-1931 and favipiravir in an expanded range. Based on the platform nature of IDentif.AI, these findings may support further explorations of the dose-dependent drug interactions between different drug classes in further pre-clinical and clinical trials as possible combinatorial therapies consisting of unmodified and nanomedicine-enabled drugs, to combat current and future COVID-19 strains and other emerging pathogens.

The IDentif.AI-x pandemic readiness platform: Rapid prioritization of optimized COVID-19 combination therapy regimens.

IDentif.AI-x, a clinically actionable artificial intelligence platform, was used to rapidly pinpoint and prioritize optimal combination therapies against COVID-19 by pairing a prospective, experimental validation of multi-drug efficacy on a SARS-CoV-2 live virus and Vero E6 assay with a quadratic optimization workflow. A starting pool of 12 candidate drugs developed in collaboration with a community of infectious disease clinicians was first narrowed down to a six-drug pool and then interrogated in 50 combination regimens at three dosing levels per drug, representing 729 possible combinations. IDentif.AI-x revealed EIDD-1931 to be a strong candidate upon which multiple drug combinations can be derived, and pinpointed a number of clinically actionable drug interactions, which were further reconfirmed in SARS-CoV-2 variants B.1.351 (Beta) and B.1.617.2 (Delta). IDentif.AI-x prioritized promising drug combinations for clinical translation and can be immediately adjusted and re-executed with a new pool of promising therapies in an actionable path towards rapidly optimizing combination therapy following pandemic emergence.

Neopterin and CXCL-10 in Cerebrospinal Fluid as Potential Biomarkers of Neuroinvasive Dengue and Chikungunya.

Dengue (DENV) and chikungunya viruses (CHIKV) cause severe neurological complications, sometimes undiagnosed. Therefore, the use of more accessible neuroinflammatory biomarkers can be advantageous considering their diagnostic and prognostic potential for aggravated clinical outcomes. In this study, we aimed to evaluate neopterin and C-X-C motif chemokine ligand 10 (CXCL-10) in cerebrospinal fluid (CSF) for the diagnosis of neuroinvasive DENV and CHIKV. We analyzed the CSF of 66 patients with neurological disorders, comprising 12 neuroinvasive DENV/CHIKV, 20 inflammatory control (viral, bacterial, and fungal meningitis, and autoimmune disorders), and 24 noninflammatory control (cerebrovascular disease, dementia, neoplasm). There was no difference between the concentration of CSF neopterin in the neuroinvasive DENV/CHIKV and control groups. However, there was a significant difference in the CXCL-10 level when comparing the neuroinvasive DENV/CHIKV group and the non-inflammatory control (p < 0.05). Furthermore, we found a linear correlation between neopterin and CXCL-10 CSF levels in the three groups. For the DENV/CHIKV neuroinvasive diagnosis, the ROC curve showed the best cut-off values for CSF neopterin at 11.23 nmol/L (sensitivity of 67% and specificity of 63%), and for CSF CXCL-10 at 156.5 pg/mL (91.7% sensitivity and specificity). These results show that CXCL-10 in CSF represents an accurate neuroinflammatory biomarker that may contribute to neuroinvasive DENV/CHIKV diagnosis.

The IDentif.AI 2.0 Pandemic Readiness Platform: Rapid Prioritization of Optimized COVID-19 Combination Therapy Regimens

ObjectivesWe aimed to harness IDentif.AI 2.0, a clinically actionable AI platform to rapidly pinpoint and prioritize optimal combination therapy regimens against COVID-19. MethodsA pool of starting candidate therapies was developed in collaboration with a community of infectious disease clinicians and included EIDD-1931 (metabolite of EIDD-2801), baricitinib, ebselen, selinexor, masitinib, nafamostat mesylate, telaprevir (VX-950), SN-38 (metabolite of irinotecan), imatinib mesylate, remdesivir, lopinavir, and ritonavir. Following the initial drug pool assessment, a focused, 6-drug pool was interrogated at 3 dosing levels per drug representing nearly 10,000 possible combination regimens. IDentif.AI 2.0 paired prospective, experimental validation of multi-drug efficacy on a SARS-CoV-2 live virus (propagated, original strain, B.1.351 and B.1.617.2 variants) and Vero E6 assay with a quadratic optimization workflow. ResultsWithin 3 weeks, IDentif.AI 2.0 realized a list of combination regimens, ranked by efficacy, for clinical go/no-go regimen recommendations. IDentif.AI 2.0 revealed EIDD-1931 to be a strong candidate upon which multiple drug combinations can be derived. ConclusionsIDentif.AI 2.0 rapidly revealed promising drug combinations for clinical translation. It pinpointed dose-dependent drug synergy behavior to play a role in trial design and realizing positive treatment outcomes. IDentif.AI 2.0 represents an actionable path towards rapidly optimizing combination therapy following pandemic emergence. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/21259321v2_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@f8a159org.highwire.dtl.DTLVardef@12908b7org.highwire.dtl.DTLVardef@fb6485org.highwire.dtl.DTLVardef@8493c3_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- When novel pathogens emerge, the immediate strategy is to repurpose drugs. - Good drugs delivered together in suboptimal combinations and doses can yield low or no efficacy, leading to misperception that the drugs are ineffective. - IDentif.AI 2.0 does not use in silico modeling or pre-existing data. - IDentif.AI 2.0 pairs optimization with prospectively acquired experimental data using a SARS-CoV-2/Vero E6 assay. - IDentif.AI 2.0 pinpoints EIDD-1931 as a foundation for optimized anti-SARS-CoV-2 combination therapies.

Remdesivir for 5 or 10 Days in Patients with Severe Covid-19.

BACKGROUND: Remdesivir is an RNA polymerase inhibitor with potent antiviral activity in vitro and efficacy in animal models of coronavirus disease 2019 (Covid-19). METHODS: We conducted a randomized, open-label, phase 3 trial involving hospitalized patients with confirmed SARS-CoV-2 infection, oxygen saturation of 94% or less while they were breathing ambient air, and radiologic evidence of pneumonia. Patients were randomly assigned in a 1:1 ratio to receive intravenous remdesivir for either 5 days or 10 days. All patients received 200 mg of remdesivir on day 1 and 100 mg once daily on subsequent days. The primary end point was clinical status on day 14, assessed on a 7-point ordinal scale. RESULTS: In total, 397 patients underwent randomization and began treatment (200 patients for 5 days and 197 for 10 days). The median duration of treatment was 5 days (interquartile range, 5 to 5) in the 5-day group and 9 days (interquartile range, 5 to 10) in the 10-day group. At baseline, patients randomly assigned to the 10-day group had significantly worse clinical status than those assigned to the 5-day group (P = 0.02). By day 14, a clinical improvement of 2 points or more on the ordinal scale occurred in 64% of patients in the 5-day group and in 54% in the 10-day group. After adjustment for baseline clinical status, patients in the 10-day group had a distribution in clinical status at day 14 that was similar to that among patients in the 5-day group (P = 0.14). The most common adverse events were nausea (9% of patients), worsening respiratory failure (8%), elevated alanine aminotransferase level (7%), and constipation (7%). CONCLUSIONS: In patients with severe Covid-19 not requiring mechanical ventilation, our trial did not show a significant difference between a 5-day course and a 10-day course of remdesivir. With no placebo control, however, the magnitude of benefit cannot be determined. (Funded by Gilead Sciences; GS-US-540-5773 ClinicalTrials.gov number, NCT04292899.).

Chemical synthesis of tripeptide thioesters for the biotechnological incorporation into the myxobacterial secondary metabolite argyrin via mutasynthesis.

The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure-activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product's biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence ᴅ-Ala-Dha-Sar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide.

Engineered Flumazenil Recognition Site Provides Mechanistic Insight Governing Benzodiazepine Modulation in GABAA Receptors.

The anxiolytic, anticonvulsant, muscle-relaxant, and sedative-hypnotic effects of benzodiazepine site ligands are mainly elicited by allosteric modulation of GABAA receptors via their extracellular αx+/γ2- ( x = 1, 2, 3, 5) interfaces. In addition, a low affinity binding site at the homologous α+/ß- interfaces was reported for some benzodiazepine site ligands. Classical benzodiazepines and pyrazoloquinolinones have been used as molecular probes to develop structure-activity relationship models for benzodiazepine site activity. Considering all possible α+/ß- and α+/γ- interfaces, such ligands potentially interact with as many as 36 interfaces, giving rise to undesired side effects. Understanding the binding modes at their binding sites will enable rational strategies to design ligands with desired selectivity profiles. Here, we compared benzodiazepine site ligand interactions in the high affinity α1+/γ2- site with the homologous α1+/ß3- site using a successive mutational approach. We incorporated key amino acids known to contribute to high affinity benzodiazepine binding of the γ2- subunit into the ß3- subunit, resulting in a quadruple mutant ß3(4mut) with high affinity flumazenil (Ro 15-1788) binding properties. Intriguingly, some benzodiazepine site ligands displayed positive allosteric modulation in the tested recombinant α1ß3(4mut) constructs while diazepam remained inactive. Consequently, we performed in silico molecular docking in the wildtype receptor and the quadruple mutant. The results led to the conclusion that different benzodiazepine site ligands seem to use distinct binding modes, rather than a common binding mode. These findings provide structural hypotheses for the future optimization of both benzodiazepine site ligands, and ligands that interact with the homologous α+/ß- sites.

SAR-Guided Scoring Function and Mutational Validation Reveal the Binding Mode of CGS-8216 at the α1+/γ2- Benzodiazepine Site.

The structural resolution of a bound ligand-receptor complex is a key asset to efficiently drive lead optimization in drug design. However, structural resolution of many drug targets still remains a challenging endeavor. In the absence of structural knowledge, scientists resort to structure-activity relationships (SARs) to promote compound development. In this study, we incorporated ligand-based knowledge to formulate a docking scoring function that evaluates binding poses for their agreement with a known SAR. We showcased this protocol by identifying the binding mode of the pyrazoloquinolinone (PQ) CGS-8216 at the benzodiazepine binding site of the GABAA receptor. Further evaluation of the final pose by molecular dynamics and free energy simulations revealed a close proximity between the pendent phenyl ring of the PQ and γ2D56, congruent with the low potency of carboxyphenyl analogues. Ultimately, we introduced the γ2D56A mutation and in fact observed a 10-fold potency increase in the carboxyphenyl analogue, providing experimental evidence in favor of our binding hypothesis.

Different Benzodiazepines Bind with Distinct Binding Modes to GABAA Receptors.

Benzodiazepines are clinically relevant drugs that bind to GABAA neurotransmitter receptors at the α+/γ2- interfaces and thereby enhance GABA-induced chloride ion flux leading to neuronal hyperpolarization. However, the structural basis of benzodiazepine interactions with their high-affinity site at GABAA receptors is controversially debated in the literature, and in silico studies led to discrepant binding mode hypotheses. In this study, computational docking of diazepam into α+/γ2- homology models suggested that a chiral methyl group, which is known to promote preferred binding to α5-containing GABAA receptors (position 3 of the seven-membered diazepine ring), could possibly provide experimental evidence that supports or contradicts the proposed binding modes. Thus, we investigated three pairs of R and S isomers of structurally different chemotypes, namely, diazepam, imidazobenzodiazepine, and triazolam derivatives. We used radioligand displacement studies as well as two-electrode voltage clamp electrophysiology in α1ß3γ2-, α2ß3γ2-, α3ß3γ2-, and α5ß3γ2-containing GABAA receptors to determine the ligand binding and functional activity of the three chemotypes. Interestingly, both imidazobenzodiazepine isomers displayed comparable binding affinities, while for the other two chemotypes, a discrepancy in binding affinities of the different isomers was observed. Specifically, the R isomers displayed a loss of binding, whereas the S isomers remained active. These findings are in accordance with the results of our in silico studies suggesting the usage of a different binding mode of imidazobenzodiazepines compared to those of the other two tested chemotypes. Hence, we conclude that different chemically related benzodiazepine ligands interact via distinct binding modes rather than by using a common binding mode.

Longitudinal Study of Cellular and Systemic Cytokine Signatures to Define the Dynamics of a Balanced Immune Environment During Disease Manifestation in Zika Virus-Infected Patients.

Background: Since its unexpected reemergence, Zika virus (ZIKV) has caused numerous outbreaks globally. This study characterized the host immune responses during ZIKV infection. Methods: Patient samples were collected longitudinally during the acute, convalescence and recovery phases of ZIKV infection over 6 months during the Singapore outbreak in late 2016. Plasma immune mediators were profiled via multiplex microbead assay, while changes in blood cell numbers were determined with immunophenotyping. Results: Data showed the involvement of various immune mediators during acute ZIKV infection accompanied by a general reduction in blood cell numbers for all immune subsets except CD14+ monocytes. Importantly, viremic patients experiencing moderate symptoms had significantly higher quantities of interferon γ-induced protein 10, monocyte chemotactic protein 1, interleukin 1 receptor antagonist, interleukin 8, and placental growth factor 1, accompanied by reduced numbers of peripheral CD8+ T cells, CD4+ T cells, and double-negative T cells. Levels of T-cell associated mediators, including interferon γ-induced protein 10, interferon γ, and interleukin 10, were high in recovery phases of ZIKV infection, suggesting a functional role for T cells. The identification of different markers at specific disease phases emphasizes the dynamics of a balanced cytokine environment in disease progression. Conclusions: This is the first comprehensive study that highlights specific cellular changes and immune signatures during ZIKV disease progression, and it provides valuable insights into ZIKV immunopathogenesis.

Towards functional selectivity for α6ß3γ2 GABAA receptors: a series of novel pyrazoloquinolinones.

BACKGROUND AND PURPOSE: The GABAA receptors are ligand-gated ion channels, which play an important role in neurotransmission. Their variety of binding sites serves as an appealing target for many clinically relevant drugs. Here, we explored the functional selectivity of modulatory effects at specific extracellular α+/ß- interfaces, using a systematically varied series of pyrazoloquinolinones. EXPERIMENTAL APPROACH: Recombinant GABAA receptors were expressed in Xenopus laevis oocytes and modulatory effects on GABA-elicited currents by the newly synthesized and reference compounds were investigated by the two-electrode voltage clamp method. KEY RESULTS: We identified a new compound which, to the best of our knowledge, shows the highest functional selectivity for positive modulation at α6ß3γ2 GABAA receptors with nearly no residual activity at the other αxß3γ2 (x = 1-5) subtypes. This modulation was independent of affinity for α+/γ- interfaces. Furthermore, we demonstrated for the first time a compound that elicits a negative modulation at specific extracellular α+/ß- interfaces. CONCLUSION AND IMPLICATIONS: These results constitute a major step towards a potential selective positive modulation of certain α6-containing GABAA receptors, which might be useful to elicit their physiological role. Furthermore, these studies pave the way towards insights into molecular principles that drive positive versus negative allosteric modulation of specific GABAA receptor isoforms.

Molecular tools for GABAA receptors: High affinity ligands for ß1-containing subtypes.

γ-Aminobutyric acid type A (GABAA) receptors are pentameric GABA-gated chloride channels that are, in mammalians, drawn from a repertoire of 19 different genes, namely α1-6, ß1-3, γ1-3, δ, ε, θ, π and ρ1-3. The existence of this wide variety of subunits as well as their diverse assembly into different subunit compositions result in miscellaneous receptor subtypes. In combination with the large number of known and putative allosteric binding sites, this leads to a highly complex pharmacology. Recently, a novel binding site at extracellular α+/ß- interfaces was described as the site of modulatory action of several pyrazoloquinolinones. In this study we report a highly potent ligand from this class of compounds with pronounced ß1-selectivity that mainly lacks α-subunit selectivity. It constitutes the most potent ß1-selective positive allosteric modulatory ligand with known binding site. In addition, a proof of concept pyrazoloquinolinone ligand lacking the additional high affinity interaction with the benzodiazepine binding site is presented. Ultimately, such ligands can be used as invaluable molecular tools for the detection of ß1-containing receptor subtypes and the investigation of their abundance and distribution.

A Sensitive Method for Detecting Zika Virus Antigen in Patients' Whole-Blood Specimens as an Alternative Diagnostic Approach.

Background: Epidemics caused by the reemergence of Zika virus (ZIKV) warrant the need to develop new diagnostic measures to complement currently used detection methods. In this study, we explored the detection of ZIKV antigen in a defined leukocyte subset from patients' whole-blood specimens. Methods: Whole-blood samples were obtained at the acute and early convalescent phases from ZIKV-infected patients during the Singapore outbreak in August-September 2016. Presence of ZIKV antigen was determined by flow cytometry staining for intracellular ZIKV NS3, using a ZIKV-specific polyclonal antibody. The presence of ZIKV antigen was determined in CD45+CD14+ monocytes. Results: Data showed that ZIKV NS3 antigen could be detected in CD45+CD14+ monocytes. The levels of detection were further categorized into 3 groups: high (positivity among >40% of monocytes), moderate (positivity among 10%-40%), and low (positivity among <10%). While a majority of patients showed a decrease in the amount of ZIKV antigen detected at later time points, some patients displayed higher levels as the disease progressed. Conclusions: Our data highlights an alternative approach in using flow cytometry as a sensitive method for detecting ZIKV antigen in whole blood. Importantly, it further confirms the role of CD14+ monocytes as an important cellular target for ZIKV infection during the viremic phase.

MRSA Transmission Dynamics Among Interconnected Acute, Intermediate-Term, and Long-Term Healthcare Facilities in Singapore.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) is the most common healthcare-associated multidrug-resistant organism. Despite the interconnectedness between acute care hospitals (ACHs) and intermediate- and long-term care facilities (ILTCFs), the transmission dynamics of MRSA between healthcare settings is not well understood. METHODS: We conducted a cross-sectional study in a network comprising an ACH and 5 closely affiliated ILTCFs in Singapore. A total of 1700 inpatients were screened for MRSA over a 6-week period in 2014. MRSA isolates underwent whole-genome sequencing, with a pairwise single-nucleotide polymorphism (Hamming distance) cutoff of 60 core genome single-nucleotide polymorphisms used to define recent transmission clusters (clades) for the 3 major clones. RESULTS: MRSA prevalence was significantly higher in intermediate-term (29.9%) and long-term (20.4%) care facilities than in the ACH (11.8%) (P < .001). The predominant clones were sequence type [ST] 22 (n = 183; 47.8%), ST45 (n = 129; 33.7%), and ST239 (n = 26; 6.8%), with greater diversity of STs in ILTCFs relative to the ACH. A large proportion of the clades in ST22 (14 of 21 clades; 67%) and ST45 (7 of 13; 54%) included inpatients from the ACH and ILTCFs. The most frequent source of the interfacility transmissions was the ACH (n = 28 transmission events; 36.4%). CONCLUSIONS: MRSA transmission dynamics between the ACH and ILTCFs were complex. The greater diversity of STs in ILTCFs suggests that the ecosystem in such settings might be more conducive for intrafacility transmission events. ST22 and ST45 have successfully established themselves in ILTCFs. The importance of interconnected infection prevention and control measures and strategies cannot be overemphasized.

Local transmission and global dissemination of New Delhi Metallo-Beta-Lactamase (NDM): a whole genome analysis.

BACKGROUND: New Delhi metallo-ß-lactamase (bla NDM), a plasmid-borne carbapenemase gene associated with significant mortality and severely limited treatment options, is of global public health concern as it is found in extremely diverse Gram-negative bacterial strains. This study thus aims to genetically characterize local and global spread of bla NDM. METHODS: To investigate local transmission patterns in the context of a single hospital, whole genome sequencing data of the first 11 bla NDM-positive bacteria isolated in a local hospital were analyzed to: (1) identify and compare bla NDM-positive plasmids; and (2) study the phylogenetic relationship of the bacteria chromosomes. The global analysis was conducted by analyzing 2749 complete plasmid sequences (including 39 bla NDM-positive plasmids) in the NCBI database, where: (1) the plasmids were clustered based on their gene composition similarity; (2) phylogenetic study was conducted for each bla NDM-positive plasmid cluster to infer the phylogenetic relationship within each cluster; (3) gene transposition events introducing bla NDM into different plasmid backbones were identified; and (4) clustering pattern was correlated with the plasmids' incompatibility group and geographical distribution. RESULTS: Analysis of the first 11 bla NDM-positive isolates from a single hospital revealed very low bla NDM-positive plasmid diversity. Local transmission was characterized by clonal spread of a predominant plasmid with 2 sporadic instances of plasmid introduction. In contrast to the low diversity locally, global bla NDM spread involved marked plasmid diversity with no predominant bacterial clone. Thirty-nine (1.4 %) out of the 2749 complete plasmid sequences were bla NDM-positive, and could be resolved into 7 clusters, which were associated with plasmid incompatibility group and geographical distribution. The bla NDM gene module was witnessed to mobilize between different plasmid backbones on at least 6 independent occasions. CONCLUSIONS: Our analysis revealed the complex genetic pathways of bla NDM spread, with global dissemination characterized mainly by transposition of the bla NDM gene cassette into varied plasmids. Early local transmission following plasmid introduction is characterized by plasmid conjugation and bacterial spread. Our findings emphasize the importance of plasmid molecular epidemiology in understanding bla NDM spread.

Psychosocial determinants of physicians' acceptance of recommendations by antibiotic computerised decision support systems: A mixed methods study.

Antibiotic computerised decision support systems (CDSSs) were developed to facilitate optimal prescribing, but acceptance of their recommendations has remained low. We aimed to evaluate physicians' perceptions and attitudes toward antibiotic CDSSs and determine psychosocial factors associated with acceptance of CDSS recommendations for empirical therapy. A mixed methods study was conducted in an adult tertiary-care hospital in Singapore, with its in-house antibiotic CDSS that integrates antimicrobial stewardship with electronic prescribing. Focus group discussions were conducted among purposively sampled physicians and data were analysed using the framework approach. Emerging themes were included in the questionnaire with newly developed scales for the subsequent cross-sectional survey involving all physicians. Principal components analysis was performed to derive the latent factor structure that was later applied in multivariate analyses. Physicians expressed confidence in the credibility of CDSS recommendations. Junior physicians accepted CDSS recommendations most of the time, whilst senior physicians acknowledged overriding recommendations in complex patients with multiple infections or allergies. Willingness to consult the CDSS for common and complex infections (OR=1.68, 95% CI 1.16-2.44) and preference for personal or team decision (OR=0.61, 95% CI 0.43-0.85) were associated with acceptance of CDSS recommendations. Cronbach's α for scales measuring physicians' attitudes and perceptions towards acceptance of CDSS recommendations ranged from 0.64 to 0.88. Physicians' willingness to consult an antibiotic CDSS determined acceptance of its recommendations. Physicians would choose to exercise their own or clinical team's decision over CDSS recommendations in complex patient situations when the antibiotic prescribing needs were not met.

Multifunctional cytomegalovirus (CMV)-specific CD8(+) T cells are not restricted by telomere-related senescence in young or old adults.

Antigen-specific multifunctional T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α simultaneously after activation are important for the control of many infections. It is unclear if these CD8(+) T cells are at an early or late stage of differentiation and whether telomere erosion restricts their replicative capacity. We developed a multi-parameter flow cytometric method for investigating the relationship between differentiation (CD45RA and CD27 surface phenotype), function (cytokine production) and replicative capacity (telomere length) in individual cytomegalovirus (CMV) antigen-specific CD8(+) T cells. This involves surface and intracellular cell staining coupled to fluorescence in situ hybridization to detect telomeres (flow-FISH). The end-stage/senescent CD8(+)  CD45RA(+)  CD27(-) T-cell subset increases significantly during ageing and this is exaggerated in CMV immune-responsive subjects. However, these end-stage cells do not have the shortest telomeres, implicating additional non-telomere-related mechanisms in inducing their senescence. The telomere lengths in total and CMV (NLV)-specific CD8(+) T cells in all four subsets defined by CD45RA and CD27 expression were significantly shorter in old compared with young individuals in both a Caucasian and an Asian cohort. Following stimulation by anti-CD3 or NLV peptide, similar proportions of triple-cytokine-producing cells are found in CD8(+) T cells at all stages of differentiation in both age groups. Furthermore, these multi-functional cells had intermediate telomere lengths compared with cells producing only one or two cytokines after activation. Therefore, global and CMV (NLV)-specific CD8(+) T cells that secrete interferon-γ, interleukin-2 and tumour necrosis factor-α are at an intermediate stage of differentiation and are not restricted by excessive telomere erosion.

Antibiotic therapy and clinical outcomes of Pseudomonas aeruginosa (PA) bacteraemia.

INTRODUCTION: Pseudomonas aeruginosa (PA) bacteraemia is associated with high morbidity and mortality. We assessed clinical outcomes in patients with PA bacteraemia treated with piperacillin-tazobactam (TZP) versus other antibiotics, and monotherapy versus combination, all with proven activity by disc testing without minimum inhibitory concentration (MIC) data. MATERIALS AND METHODS: All patients with PA bacteraemia in 2007 to 2008 were reviewed for demographic, comorbidity, clinical, laboratory, treatment and outcome data. Primary outcome was 30-day mortality. Secondary outcomes included microbiological clearance, clinical response and length of stay (LOS). RESULTS: Median age for 91 patients was 65 years. Median Simplified Acute Physiology Score (SAPS) II score was 30. Monotherapy was used in 77 cases: 42 on ceftazidime, 17 on TZP, 10 on carbapenems, and 8 on other antipseudomonal antibiotics. The 30-day mortality was 20.9%, and similar between ceftazidime and TZP versus other antibiotics respectively. More patients in combination versus monotherapy group had cardiovascular diseases, diabetes mellitus and vascular access as source of bacteraemia. Patients on monotherapy had higher 30-day mortality (24.7% vs 0%, P = 0.037). Multivariate analysis identified SAPS II score (OR = 1.097, 95% CI, 1.032 to 1.166, P = 0.003) and cancer (OR = 4.873, 95% CI, 1.235 to 19.223, P = 0.024) as independent predictors of 30-day mortality. CONCLUSION: TZP appeared to be an effective culture-guided antibiotic for PA bacteraemia. High 30-day mortality in monotherapy might be confounded by comorbidity, illness severity and sample size. Cancer patients and a high SAPS II score were independent predictors of 30-day mortality.

Antibiotic therapy and clinical outcomes of Pseudomonas aeruginosa (PA) bacteraemia

<p><b>INTRODUCTION</b>Pseudomonas aeruginosa (PA) bacteraemia is associated with high morbidity and mortality. We assessed clinical outcomes in patients with PA bacteraemia treated with piperacillin-tazobactam (TZP) versus other antibiotics, and monotherapy versus combination, all with proven activity by disc testing without minimum inhibitory concentration (MIC) data.</p><p><b>MATERIALS AND METHODS</b>All patients with PA bacteraemia in 2007 to 2008 were reviewed for demographic, comorbidity, clinical, laboratory, treatment and outcome data. Primary outcome was 30-day mortality. Secondary outcomes included microbiological clearance, clinical response and length of stay (LOS).</p><p><b>RESULTS</b>Median age for 91 patients was 65 years. Median Simplified Acute Physiology Score (SAPS) II score was 30. Monotherapy was used in 77 cases: 42 on ceftazidime, 17 on TZP, 10 on carbapenems, and 8 on other antipseudomonal antibiotics. The 30-day mortality was 20.9%, and similar between ceftazidime and TZP versus other antibiotics respectively. More patients in combination versus monotherapy group had cardiovascular diseases, diabetes mellitus and vascular access as source of bacteraemia. Patients on monotherapy had higher 30-day mortality (24.7% vs 0%, P = 0.037). Multivariate analysis identified SAPS II score (OR = 1.097, 95% CI, 1.032 to 1.166, P = 0.003) and cancer (OR = 4.873, 95% CI, 1.235 to 19.223, P = 0.024) as independent predictors of 30-day mortality.</p><p><b>CONCLUSION</b>TZP appeared to be an effective culture-guided antibiotic for PA bacteraemia. High 30-day mortality in monotherapy might be confounded by comorbidity, illness severity and sample size. Cancer patients and a high SAPS II score were independent predictors of 30-day mortality.</p>

Severe community-acquired Acinetobacter baumannii pneumonia: an emerging highly lethal infectious disease in the Asia-Pacific.

BACKGROUND AND OBJECTIVE: Patients with community-acquired Acinetobacter baumannii (AB) pneumonia have been reported from subtropical countries. We investigated the epidemiology, clinical and microbiological characteristics of community-acquired pneumonia (CAP) due to AB in Singapore. METHOD: A retrospective case series was performed over a 21-month period at two institutions. RESULTS: From 1 January 2007 to 30 September 2008, eight patients were diagnosed with CAP due to AB. Seven had bacteraemia and five were sputum culture-positive. The median age at presentation was 58.5 years (range 45-76 years). Five patients (71.4%) acquired the pneumonia in the warmer months of June to September. Presentation was acute, with a median duration of 2.5 days (range 1-7 days). The median Acute Physiology and Chronic Health Evaluation II score was 28.5 (range 6-36). Six patients presented with septic shock, lactic acidosis, acute kidney injury and respiratory failure, necessitating ICU care; five of these patients eventually died. All patients received empirical antibiotics, including third-generation cephalosporins, which were inactive against the organism. All isolates were susceptible to ampicillin/sulbactam, ciprofloxacin, co-trimoxazole, aminoglycosides and imipenem. CONCLUSIONS: Community-acquired AB pneumonia have a fulminant course. In a region endemic for melioidosis and severe community-acquired Klebsiella pneumoniae, the challenge lies in rapid identification and initiation of appropriate empirical antibiotics to improve the survival of patients with AB CAP.