Post-COVID care delivery: The experience from an Irish tertiary centre's post-COVID clinic.

BACKGROUND: The long-term effects of SARS-CoV-2 infection and optimal follow-up approach are not well-recognised. Here we describe the implementation of a post-COVID clinic in an Irish tertiary centre after the first wave of the pandemic. This study describes the characteristics of our patient cohort and the operations and outcomes of the clinic, exploring some of the risk factors for developing post-COVID syndrome and the appropriateness of the triage system employed. METHODS: All SARS-CoV-2 positive patients from March 10th to June 14th 2020 were telephone-triaged as red, amber or green based on ongoing symptoms with clinic appointments scheduled accordingly. All clinic visits were face-to-face with the infectious diseases medical team and a proforma for each patient was completed. Data were collected retrospectively by reviewing the proformas and the electronic medical record (EMR). RESULTS: 311 patients attended the clinic. Median time from illness to clinic appointment was 95 days (IQR 77-105.5). 204 patients (66%) were female, 192 (62%) were hospital staff, and the median age was 43 years (IQR 31-53). 138 patients (44%) had required hospital admission. At their first clinic visit 219 patients (70%) had ongoing symptoms. A further appointment was made for 62 patients (20%). 34 patients (11%) were discussed at an MDT meeting, and 55 (18%) were referred onward to a specialist service. 85% of those triaged green, 73% of those triaged amber, and 39% of those triaged red did not receive further follow up after one clinic visit. Patients were more likely to require follow up with reported dyspnoea (OR 5.6; 95% CI 2.8-11.3; p <0.001), cough (OR 3.0; 95% CI 1.1-8.4, p = 0.04), and palpitations (OR 3.6; 95% CI 1.0-12.3; p = 0.04). Female sex was associated with increased odds of a higher triage category (OR 1.8; 95% CI 1.08 to 3.20; p = 0.02), as was requiring admission to hospital (OR 4.0; 95% CI 2.34 to 6.90; p < 0.001). CONCLUSION: The long-term effects of COVID-19 are significant with 70% of our cohort experiencing persistent symptoms. Persistent dyspnoea, cough and palpitations were associated with increased need for follow up. This study also suggests that a traffic light telephone-triage service followed by a face-to-face medical-led clinic could be an effective way of identifying patients who require further management.

Histamine Produced by Gram-Negative Bacteria Impairs Neutrophil's Antimicrobial Response by Engaging the Histamine 2 Receptor.

We found that histamine (10-9 M) did not have any effect on the in vitro capture of Escherichia coli by neutrophils but accelerated its intracellular killing. In contrast, histamine (10-6 M) delayed the capture of Escherichia coli by neutrophils and reduced the amounts of pHrodo zymosan particles inside acidic mature phagosomes. Histamine acted through the H4R and the H2R, which are coupled to the Src family tyrosine kinases or the cAMP/protein kinase A pathway, respectively. The protein kinase A inhibitor H-89 abrogated the delay in bacterial capture induced by histamine (10-6 M) and the Src family tyrosine kinase inhibitor PP2 blocked histamine (10-9 M) induced acceleration of bacterial intracellular killing and tyrosine phosphorylation of proteins. To investigate the role of histamine in pathogenicity, we designed an Acinetobacter baumannii strain deficient in histamine production (hdc::TOPO). Galleria mellonella larvae inoculated with the wild-type Acinetobacter baumannii ATCC 17978 strain (1.1 × 105 CFU) died rapidly (100% death within 40 h) but not when inoculated with the Acinetobacter baumannii hdc::TOPO mutant (10% mortality). The concentration of histamine rose in the larval haemolymph upon inoculation of the wild type but not the Acinetobacter baumannii hdc::TOPO mutant, such concentration of histamine blocks the ability of hemocytes from Galleria mellonella to capture Candida albicans in vitro. Thus, bacteria-producing histamine, by maintaining high levels of histamine, may impair neutrophil phagocytosis by hijacking the H2R.

Galleria mellonella: The Versatile Host for Drug Discovery, In Vivo Toxicity Testing and Characterising Host-Pathogen Interactions.

Larvae of the greater wax moth, Galleria mellonella, are a convenient in vivo model for assessing the activity and toxicity of antimicrobial agents and for studying the immune response to pathogens and provide results similar to those from mammals. G. mellonella larvae are now widely used in academia and industry and their use can assist in the identification and evaluation of novel antimicrobial agents. Galleria larvae are inexpensive to purchase and house, easy to inoculate, generate results within 24-48 h and their use is not restricted by legal or ethical considerations. This review will highlight how Galleria larvae can be used to assess the efficacy of novel antimicrobial therapies (photodynamic therapy, phage therapy, metal-based drugs, triazole-amino acid hybrids) and for determining the in vivo toxicity of compounds (e.g., food preservatives, ionic liquids) and/or solvents (polysorbate 80). In addition, the disease development processes are associated with a variety of pathogens (e.g., Staphylococcus aureus, Listeria monocytogenes, Aspergillus fumigatus, Madurella mycotomatis) in mammals are also present in Galleria larvae thus providing a simple in vivo model for characterising disease progression. The use of Galleria larvae offers many advantages and can lead to an acceleration in the development of novel antimicrobials and may be a prerequisite to mammalian testing.

Multi-targeted metallo-ciprofloxacin derivatives rationally designed and developed to overcome antimicrobial resistance.

Antimicrobial resistance is a major global threat to human health due to the rise, spread and persistence of multi-drug-resistant bacteria or 'superbugs'. There is an urgent need to develop novel chemotherapeutics to overcome this overarching challenge. The authors derivatized a clinically used fluoroquinolone antibiotic ciprofloxacin (Cip), and complexed it to a copper phenanthrene framework. This resulted in the development of two novel metallo-antibiotics of general formula [Cu(N,N)(CipHA)]NO3 where N,N represents a phenanthrene ligand and CipHA represents a hydroxamic acid of Cip derivative. Comprehensive studies, including a detailed proteomic study in which Staphylococcus aureus cells were exposed to the complexes, were undertaken to gain an insight into their mode of action. These new complexes possess potent antibacterial activity against S. aureus and methicillin-resistant S. aureus. In addition, they were found to be well tolerated in vivo in Galleria mellonella larvae, which has both functional and structural similarities to the innate immune system of mammals. These findings suggest that proteins involved in virulence, pathogenesis, and the synthesis of nucleotides and DNA repair mechanisms are most affected. In addition, both complexes affected similar cell pathways when compared with clinically used Cip, including cationic antimicrobial peptide resistance. The Cu-DPPZ-CipHA (DPPZ = dipyrido[3,2-a:2',3'-c]phenazine) analogue also induces cell leakage, which leads to an altered proteome indicative of reduced virulence and increased stress.

Rapid and Laboratory SARS-CoV-2 Antibody Testing in High-Risk Hospital Associated Cohorts of Unknown COVID-19 Exposure, a Validation and Epidemiological Study After the First Wave of the Pandemic.

Objective: We aimed to use SARS-CoV-2 antibody tests to assess the asymptomatic seroprevalence of individuals in high-risk hospital cohorts who's previous COVID-19 exposure is unknown; staff, and patients requiring haemodialysis or chemotherapy after the first wave. Methods: In a single Center, study participants had five SARS-CoV-2 antibody tests done simultaneously; one rapid diagnostic test (RDT) (Superbio Colloidal Gold IgM/IgG), and four laboratory tests (Roche Elecsys® Anti-SARS-CoV-2 IgG [RE], Abbott Architect i2000SR IgG [AAr], Abbott Alinity IgG [AAl], and Abbott Architect IgM CMIA). To determine seroprevalence, only positive test results on laboratory assay were considered true positives. Results: There were 157 participants, of whom 103 (65.6%) were female with a median age of 50 years (range 19-90). The IgG component of the RDT showed a high number of false positives (n = 18), was inferior to the laboratory assays (p < 0.001 RDT vs. AAl/AAr, p < 0.001 RDT vs. RE), and had reduced specificity (85.5% vs. AAl/AAr, 87.2% vs. RE). Sero-concordance was 97.5% between IgG laboratory assays (RE vs. AAl/AAr). Specificity of the IgM component of the RDT compared to Abbott IgM CMIA was 95.4%. Ten participants had positivity in at least one laboratory assay, seven (9.9%) of which were seen in HCWs. Two (4.1%) hematology/oncology (H/O) patients and a single (2.7%) haemodialysis (HD) were asymptomatically seropositive. Asymptomatic seroprevalence of HCWs compared to patients was not significant (p = 0.105). Conclusion: HCWs (9.9%) had higher, although non-significant asymptomatic seroprevalence of SARS-CoV-2 antibodies compared to high-risk patients (H/O 4.1%, HD 2.7%). An IgM/IgG rapid diagnostic test was inferior to laboratory assays. Sero-concordance of 97.5% was found between IgG laboratory assays, RE vs. AAl/AAr.

False detection of rifampicin resistance using Xpert® MTB/RIF Ultra assay due to an A451V mutation in Mycobacterium tuberculosis.

BACKGROUND: In a 12 month period, three Irish-born adult cases with pulmonary TB were initially diagnosed by Xpert® MTB/RIF Ultra assay, which detected a rifampicin resistance-conferring mutation prompting treatment as potential MDR cases. METHODS: Further laboratory investigations on the cultured isolates included GenoType MTBDRplus assay, phenotypic drug susceptibility tests using the BD BACTEC MGIT culture system and MIC broth microdilution tests. Sequencing of the rpoB gene was performed using Sanger sequencing and WGS. RESULTS: Phenotypic drug susceptibility tests determined the isolates to be rifampicin susceptible. Molecular investigations identified an A451V (codon 532) mutation in the Mycobacterium tuberculosis rpoB gene that has not previously been found to cause rifampicin resistance. Genome sequencing revealed that the three isolates' genomes differed by ≤5 SNPs, indicating a high likelihood of recent transmission events. Furthermore, a cluster of six related M. tuberculosis isolates from our in-house typing database showed four were highly related; all were rifampicin susceptible and lacked this mutation. CONCLUSIONS: False detection of rifampicin resistance, albeit rare, should be considered possible with Xpert® MTB/RIF Ultra assay, particularly in low TB incidence settings. Confirmatory sequencing methods should be performed to prevent the unnecessary use of second-line anti-tuberculous drugs.

Antibiotic prescribing patterns in patients hospitalized with COVID-19: lessons from the first wave.

BACKGROUND: A high proportion of hospitalized patients with COVID-19 receive antibiotics despite evidence to show low levels of true bacterial coinfection. METHODS: A retrospective cohort study examining antibiotic prescribing patterns of 300 patients sequentially diagnosed with COVID-19. Patients were grouped into 3 sub-cohorts: Group 1 received no antibiotics, Group 2 received antibiotics for microbiologically confirmed infections and Group 3 was empirically treated with antibiotics for pneumonia. The primary aim was to identify factors that influenced prescription and continuation of antibiotics in Group 3. Secondary aims were to examine differences in outcomes between groups. RESULTS: In total, 292 patients were included (63 Group 1, 35 Group 2, 194 Group 3), median age was 60 years (IQR 44-76) and the majority were ethnically Irish (62%). The median duration of antibiotics was 7 days (IQR 5-10). In Group 3, factors associated with prescription IV antibiotics on admission were raised C-reactive protein (CRP) (P = 0.024), increased age (P = 0.023), higher quick SOFA (P = 0.016) score and fever >37.5 °C (P = 0.011). Factors associated with duration of antibiotic course were duration of hypoxia (P < 0.001) and maximum respiratory support requirement (P = 0.013). Twenty-one patients in Group 3 had one or more antibiotic escalation events, most (n = 139) had no escalation or de-escalation of therapy. CONCLUSIONS: Duration of hypoxia and need for respiratory support may have acted as surrogate measures of improvement where usual response measures (CRP, neutrophilia, culture clearance) were absent. Continuous review of antibiotic prescriptions should be at the forefront of clinical management of hospitalized patients with COVID-19.

Documentation of Do-Not-Attempt-Cardiopulmonary-Resuscitation orders amid the COVID-19 pandemic.

INTRODUCTION: the COVID-19 pandemic has brought the decision-making process regarding cardiopulmonary resuscitation (CPR) into focus. The aim of this study is to compare rates of Do-Not-Attempt-CPR (DNACPR) documentation in older hospitalised patients before and during the COVID-19 pandemic. METHODS: this was a retrospective repeated cross-sectional study. Data including co-morbidities and resuscitation status was collected on 300 patients with COVID-19 hospitalised from 1 March to 31 May 2020. DNACPR documentation rates in patients aged ≥65 years with a diagnosis of COVID-19 were compared to those without COVID-19 admitted during the same period and were also compared to the documentation rates pre-COVID-19 pandemic (1 March-31 May 2019). RESULTS: of 300 COVID-19-positive patients, 28% had a DNACPR order documented during their admission. Of 131 older (≥65 years) patients with COVID-19, 60.3% had a DNACPR order compared to 25.4% of 130 older patients without COVID-19 (P < 0.0001). During a comparable time period pre-pandemic, 15.4% of 130 older patients had a DNACPR order in place (P < 0.0001). Almost fifty percent of DNACPR orders were recorded within 24 h of a positive swab result for SARS-CoV-2. Of older COVID-19-positive patients, 39.2% were referred to palliative care services and 70.2% survived. CONCLUSION: the COVID-19 pandemic has prompted more widespread and earlier decision-making regarding resuscitation status. Although case fatality rates were higher for older hospitalised patients with COVID-19, many older patients survived the illness. Advance care planning should be prioritised in all patients and should remain as part of good clinical practice despite the pandemic.

Proteomic profiling of bacterial and fungal induced immune priming in Galleria mellonella larvae.

Some insects display immunological priming as a result of elevated humoral and cellular responses which give enhanced survival against subsequent infection. The humoral immune response of Galleria mellonella larvae following pre-exposure to heat killed Staphylococcus aureus or Candida albicans cells was determined by quantitative mass spectrometry in order to assess the relationship between the humoral immune response and resistance to subsequent bacterial or fungal infection. Larvae pre-exposed to heat killed S. aureus showed increased resistance to subsequent bacterial and fungal infection. Larvae displayed an increased hemocyte density (14.08 ± 2.14 × 106 larva-1 (p < 0.05) compared to the PBS injected control [10.41 ± 1.67 × 106 larva-1]) and increased abundance of antimicrobial proteins (cecropin-D-like peptide (+22.23 fold), hdd11 (+12.61 fold) and prophenol oxidase activating enzyme 3 (+5.96 fold) in response to heat killed S. aureus. Larvae pre-exposed to heat killed C. albicans cells were resistant to subsequent fungal infection but not bacterial infection and showed a reduced hemocyte density (6.01 ± 1.63 × 106 larva-1 (p < 0.01) and increased abundance of hdd11 (+32.73 fold) and moricin-like peptide C1 (+16.76 fold). While immune priming is well recognised in G. mellonella larvae the results presented here indicate distinct differences in the response of larvae following exposure to heat killed bacterial and fungal cells.

Innate Immune Responses of Galleria mellonella to Mycobacterium bovis BCG Challenge Identified Using Proteomic and Molecular Approaches.

The larvae of the insect Galleria mellonella, have recently been established as a non-mammalian infection model for the Mycobacterium tuberculosis complex (MTBC). To gain further insight into the potential of this model, we applied proteomic (label-free quantification) and transcriptomic (gene expression) approaches to characterise the innate immune response of G. mellonella to infection with Mycobacterium bovis BCG lux over a 168 h time course. Proteomic analysis of the haemolymph from infected larvae revealed distinct changes in the proteome at all time points (4, 48, 168 h). Reverse transcriptase quantitative PCR confirmed induction of five genes (gloverin, cecropin, IMPI, hemolin, and Hdd11), which encoded proteins found to be differentially abundant from the proteomic analysis. However, the trend between gene expression and protein abundance were largely inconsistent (20%). Overall, the data are in agreement with previous phenotypic observations such as haemocyte internalization of mycobacterial bacilli (hemolin/ß-actin), formation of granuloma-like structures (Hdd11), and melanization (phenoloxidase activating enzyme 3 and serpins). Furthermore, similarities in immune expression in G. mellonella, mouse, zebrafish and in vitro cell-line models of tuberculosis infection were also identified for the mechanism of phagocytosis (ß-actin). Cecropins (antimicrobial peptides), which share the same α-helical motif as a highly potent peptide expressed in humans (h-CAP-18), were induced in G. mellonella in response to infection, giving insight into a potential starting point for novel antimycobacterial agents. We believe that these novel insights into the innate immune response further contribute to the validation of this cost-effective and ethically acceptable insect model to study members of the MTBC.

UtilisingGalleria mellonella larvae for studying in vivo activity of conventional and novel antimicrobial agents.

The immune response of insects displays many structural and functional similarities to the innate immune response of mammals. As a result of these conserved features, insects may be used for evaluating microbial virulence or for testing the in vivo efficacy and toxicity of antimicrobial compounds and results show strong similarities to those from mammals. Galleria mellonella larvae are widely used in this capacity and have the advantage of being easy to use, inexpensive to purchase and house, and being free from the ethical and legal restrictions that relate to the use of mammals in these tests. Galleria mellonella larvae may be used to assess the in vivo toxicity and efficacy of novel antimicrobial compounds. A wide range of antibacterial and antifungal therapies have been evaluated in G. mellonella larvae and results have informed subsequent experiments in mammals. While insect larvae are a convenient and reproducible model to use, care must be taken in their use to ensure accuracy of results. The objective of this review is to provide a comprehensive account of the use of G. mellonella larvae for assessing the in vivo toxicity and efficacy of a wide range of antibacterial and antifungal agents.

Zinc Chelators as Carbapenem Adjuvants for Metallo-ß-Lactamase-Producing Bacteria: In Vitro and In Vivo Evaluation.

Infections caused by metallo-ß-lactamase (MBL)-producing bacteria are emerging and carry a significant impact on patients' outcome. MBL producers are spread worldwide, both in community and hospital setting, with increasingly reported epidemic clusters and the search for MBL inhibitors is an important topic for public health. MBLs are zinc-dependent enzymes whose functioning can be hampered by zinc chelators. We evaluated the potential of six zinc chelators (disulfiram, nitroxoline, 5-amino-8-hydroxyquinoline, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid [DOTA], cyclam, and N,N,N',N'-tetrakis (2-pyridymethyl) ethylenediamine [TPEN]) in restoring carbapenem activity against MBL producers. Zinc chelators alone or in combination with meropenem against MBL-producing Klebsiella pneumoniae, Chryseobacterium indologenes, Elizabethkingia meningoseptica, and Stenotrophomonas maltophilia isolates were tested in vitro and in vivo (Galleria mellonella). In vitro experiments showed a synergistic activity between TPEN and meropenem toward all the strains. Nitroxoline alone retained activity against S. maltophilia, C. indologenes, and E. meningoseptica. In vivo experiments showed that TPEN or nitroxoline in combination with meropenem increased survival in larvae infected with E. meningoseptica, S. maltophilia, and K. pneumoniae. Based on our data, zinc chelators are potential carbapenem adjuvants molecules (restoring carbapenem activity) against MBL-sustained infections and could represent an interesting option for infections induced by these microorganisms.

Investigating the effect of antiretroviral switch to tenofovir alafenamide on lipid profiles in people living with HIV.

BACKGROUND: Whilst reporting improved renal and bone safety profiles, studies have noted changes in lipid profiles among people living with HIV (PLWH) receiving antiretroviral therapy (ART) switching away from tenofovir disoproxil fumarate (TDF) to tenofovir alafenamide (TAF). We aimed to characterize changes in lipids observed after switching to TAF-containing ART in a real-world setting. METHODS: A prospective study on PLWH enrolled in the UCD-ID Cohort study who switched to TAF-containing ART. Routine laboratory data [including lipids (total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL) and triglycerides], ART history and use of lipid-lowering therapy (LLT) were analysed preswitch and postswitch to TAF. Dyslipidaemia was classified according to the National Cholesterol Education Program-Adult Panel III (NCEP-ATP III). Change in lipid parameters and change in the proportion of individuals with dyslipidaemia postswitch was assessed using the paired t-test and the Stuart--Maxwell test, respectively. RESULTS: Of 775 PLWH enrolled in the cohort, 238 switched to TAF containing ART, of whom 194 had both preswitch and postswitch lipids measured a median (IQR) 24 (14-41) weeks postswitch to TAF. TC, LDL, HDL, triglycerides and TC : HDL ratio significantly increased postswitch [mean change (SE) mmol/l; +0.37 (0.06), P < 0.001; +0.25 (0.06), P < 0.001; +0.05 (0.02), P = 0.003, +0.13 (0.07), P = 0.02, and +0.16 (0.08), P = 0.013) respectively]. There were significant increases in the proportions of PLWH with more severe dyslipidaemia postswitch across TC and LDL (both P < 0.001). CONCLUSION: These data suggest clinically relevant, worsening lipid profiles postswitch to TAF, with a larger proportion of PLWH exceeding recommended lipid thresholds postswitch. How these changes will impact on cardiovascular risk or need for LLT remains to be determined.

Interferon lambda rs368234815 ΔG/ΔG is associated with higher CD4+:CD8+ T-cell ratio in treated HIV-1 infection.

BACKGROUND: The objectives of this study were to investigate the relationships between polymorphisms at the interferon lambda (IFNL) locus and CD4+:CD8+ ratio normalisation in people living with HIV (PLWH) on effective antiretroviral therapy (ART); and to examine whether these polymorphisms influence the composition of T lymphocyte compartments in long-term treated HIV-1 infection. METHODS: A cross-sectional study in PLWH enrolled into the Mater Immunology study. We performed IFNL genotyping on stored samples and evaluated the association of IFNL single-nucleotide polymorphisms (rs368234815 and rs12979860) with CD4+:CD8+ ratio normalization (> 1) and expanded CD4+ and CD8+ T-cell subsets; CD45RO+CD62L+ (central-memory), CD45RO+ CD62L-(effector-memory) and CD45RO-CD62L+ (naïve), using logistic and linear regression models, respectively. RESULTS: 190 ambulatory PLWH recruited to the main study, 143 were included in the analysis (38 had no stored DNA and 9 no T-lymphocyte subpopulation). Of 143 included, the median age (IQR) was 45(39-48) years, 64% were male and 66% were of Caucasian ethnicity. Heterosexual-contact (36%), injecting drug-use (33%) and men who have sex with men (24%) were the most presented HIV-transmission risk groups. The majority of subjects (90.2%) were on ART with 79% of the cohort having an undetectable HIV-RNA (< 40 copies/ml) and the time since ART initiation was 7.5 (3.7-10.4) year. rs368234815 and rs12979860 displayed similar allelic frequencies, with minor alleles ΔG and T representing 39% and 42%, respectively, of circulating alleles. rs368234815 ΔG/ΔG minor homozygotes were significantly associated with increased odds for attaining a normalised CD4+:CD8+ ratio compared to rs368234815 T/T major homozygotes in PLWH virologically suppressed on effective ART (OR = 3.11; 95% CI [1.01:9.56]). rs368234815 ΔG/ΔG homozygosity was also significantly associated with lower levels of CD4+ effector memory T-cells (regression coefficient: - 7.1%, p = 0.04) and CD8+ naïve T-cell subsets were significantly higher in HIV-1 mono-infected PLWH with rs368234815 ΔG/ΔG (regression coefficient: + 7.2%, p = 0.04). CONCLUSIONS: In virally-suppressed, long-term ART-treated PLWH, rs368234815 ΔG/ΔG homozygotes were more likely to have attained normalisation of their CD4+:CD8+ ratio, displayed lower CD4+ effector memory and higher naive CD8+ T-cells. Further studies are needed to replicate our findings in other, larger and more diverse cohorts and to determine the impact of IFNL genetic-variation on CD4+:CD8+ ratio normalisation and clinical outcomes in PLWH.

Exposure of Aspergillus fumigatus to Atorvastatin Leads to Altered Membrane Permeability and Induction of an Oxidative Stress Response.

Aspergillus fumigatus is a serious cause of disease in immune-deficient patients and in those with pulmonary malfunction (e.g., cystic fibrosis (CF), asthma). Atorvastatin is a member of the statin drug family, which are the main therapeutic agents used to decrease high serum cholesterol levels by inhibiting (HMG-CoA) reductase enzyme. The aim of the work presented here was to analyse the antifungal activity of atorvastatin and assess its effect on the virulence of A. fumigatus. Atorvastatin demonstrated strong antifungal activity and reduced the growth and viability of A. fumigatus. Exposure of A. fumigatus to atorvastatin led to a reduction in ergosterol content and increased membrane permeability, as evidenced by the release of protein, amino acids and gliotoxin. Proteomic analysis revealed an increased abundance of proteins associated with an oxidative stress response, such as the glutathione s-transferase family protein (+8.43-fold), heat shock protein Hsp30/Hsp42 (+2.02-fold) and 5-demethoxyubiquinone hydroxylase, mitochondrial (+1.73-fold), as well as secondary metabolites such as isocyanide synthase A icsA (+8.52-fold) and non-ribosomal peptide synthetase fmpE (+3.06-fold). The results presented here indicate that atorvastatin has strong antifungal properties and may have potential application in the treatment of A. fumigatus infections alone or in combination with existing antifungal agents.

Proteomic analysis of the processes leading to Madurella mycetomatis grain formation in Galleria mellonella larvae.

Mycetoma is a neglected chronic and granulomatous infection primarily associated with the fungal pathogen Madurella mycetomatis. Characteristic of this infection is the formation of grains. However, the processes leading to grain formation are not known. In this study, we employed a proteomic approach to characterise M. mycetomatis grain formation in Galleria mellonella larvae and map the processes leading to grain formation over time. For this, at 1 day, 3 days and 7 days post-inoculation, proteins from grains and hemolymph were extracted and analysed by label-free mass spectrometry. A total of 87, 51 and 48 M. mycetomatis proteins and 713, 997, 18 G. mellonella proteins were found in grains on day 1, 3 and 7 post-inoculation respectively. M. mycetomatis proteins were mainly involved in cellular metabolic processes and numerous enzymes were encountered. G. mellonella proteins were primarily involved in the nodulation process. The proteins identified were linked to nodulation and grain formation and four steps of grain formation were identified. The results of this proteomic approach could in the future be used to design novel strategies to interfere with mycetoma grain formation and to combat this difficult to treat infection.

Immune priming: the secret weapon of the insect world.

Insects are a highly successful group of animals that inhabit almost every habitat and environment on Earth. Part of their success is due to a rapid and highly effective immune response that identifies, inactivates, and eliminates pathogens. Insects possess an immune system that shows many similarities to the innate immune system of vertebrates, but they do not possess an equivalent system to the antibody-mediated adaptive immune response of vertebrates. However, some insect do display a process known as immune priming in which prior exposure to a sublethal dose of a pathogen, or pathogen-derived material, leads to an elevation in the immune response rendering the insect resistant to a subsequent lethal infection a short time later. This process is mediated by an increase in the density of circulating hemocytes and increased production of antimicrobial peptides. Immune priming is an important survival strategy for certain insects while other insects that do not show this response may have colony-level behaviors that may serve to limit the success of pathogens. Insects are now widely used as in vivo models for studying microbial pathogens of humans and for assessing the in vivo efficacy of antimicrobial agents. Knowledge of the process of immune priming in insects is essential in these applications as it may operate and augment the perceived in vivo antimicrobial activity of novel compounds.Abbreviations: 1,3-dibenzyl-4,5-diphenyl-imidazol-2-ylidene silver(I) acetate; SBC3: antimicrobial peptides; AMPs: dorsal-related immunity factor; DIF: Down syndrome cell adhesion molecule; Dscam: Lipopolysaccharide; LPS: Pathogen-associated molecular patterns; PAMPS: Patterns recognition receptors; PRR: Prophenoloxidase; PO: Toll-like receptors; TLRs: Toll/IL-1R; TIR, Transgenerational Immune Priming; TgIP: Tumor necrosis factor-α; TNF-α.

Candida albicans increases the pathogenicity of Staphylococcus aureus during polymicrobial infection of Galleria mellonella larvae.

This study detailed the responses of Galleria mellonella larvae to disseminated infection caused by co-infection with Candida albicans and Staphylococcus aureus. Doses of C. albicans (1×105 larva-1) and S. aureus (1×104 larva-1) were non-lethal in mono-infection but when combined significantly (P<0.05) reduced larval survival at 24, 48 and 72 h relative to larvae receiving S. aureus (2×104 larva-1) alone. Co-infected larvae displayed a significantly higher density of S. aureus larva-1 compared to larvae infected solely with S. aureus. Co-infection resulted in dissemination throughout the host and the appearance of large nodules. Co-infection of larvae with C. albicans and S. aureus (2×104 larva-1) resulted in an increase in the density of circulating haemocytes compared to that in larvae infected with only S. aureus. Proteomic analysis of co-infected larval haemolymph revealed increased abundance of proteins associated with immune responses to bacterial and fungal infection such as cecropin-A (+45.4-fold), recognition proteins [e.g. peptidoglycan-recognition protein LB (+14-fold)] and proteins associated with nodule formation [e.g. Hdd11 (+33.3-fold)]. A range of proteins were also decreased in abundance following co-infection, including apolipophorin (-62.4-fold), alpha-esterase 45 (-7.7-fold) and serine proteinase (-6.2-fold). Co-infection of larvae resulted in enhanced proliferation of S. aureus compared to mono-infection and an immune response showing many similarities to the innate immune response of mammals to infection. The utility of G. mellonella larvae for studying polymicrobial infection is highlighted.